Anti-fcrh5 antibodies

ABSTRACT

The invention provides anti-FcRH5 antibodies and immunoconjugates and methods of using the same.

RELATED APPLICATIONS

The present application claims benefit under 35 U.S.C. §119 of U.S.Provisional Patent Application No. 61/838,534, filed on Jun. 24, 2013,the disclosure of which is hereby incorporated by reference in itsentirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. The ASCII text file was created on Jun. 9,2014, is named GNE-0413US_SL.txt and is 67,909 bytes in size.

FIELD OF THE INVENTION

Provided herein are anti-FcRH5 antibodies (e.g., bispecific antibodies)and immunoconjugates and methods of using the same.

BACKGROUND

The Fc receptor-like 5 (FcRL5, also known as FcRH5 and IRTA2) belongs toa family of 6 recently identified genes of the immunoglobulinsuperfamily (IgSF). This family of genes is closely related to the Fcreceptors with the conserved genomic structure, extracellular Ig domaincomposition and the ITIM- and ITAM-like signaling motifs (Davis R S etal., Eur J Immunol (2005) 35:674-80). Members of this family have alsobeen called IFGPs (from Ig super-family, FcR, gp42) and SPAPs (SH2domain-containing phosphatases anchor proteins). Six members of theFcRH/IRTA receptor family have been described: FcRH1/IRTA5, FcRH2/IRTA4,FcRH3/IRTA3, FcRH4/IRTA1, FcRH5/IRTA2 and FcRH6 (Polson A G et al., Int.Immunol. (2006) 18(9):1363-1373). All FcRH/IRTAs contain somecombination of canonical immunoreceptor tyrosine-based inhibitory motifsand ‘immunoreceptor tyrosine-based activation motifs-like’ signalingmotifs. The FcRH cDNAs encode type I transmembrane glycoproteins withmultiple Ig-like extracellular domains and cytoplasmic domainscontaining consensus immunoreceptor tyrosine-based activating and/orinhibitory signaling motifs. The FcRH genes are structurally related,and their protein products share 28-60% extracellular identity with eachother. They also share 15-31% identity with their closest FcR relatives.There is a high degree of homology between the different FcRHs.

The ligand(s) for FcRH5 are unknown, but FcRH5 has been implicated inenhanced proliferation and downstream isotype expression during thedevelopment of antigen-primed B-cells (Dement-Brown J. et al. J LeukocBiol (2012) 91:59-67). The FcRH5 locus has three major mRNA isoforms(FcRH5a, FcRH5b, and FcRH5c). The major FcRH5 protein isoforms encodedby these transcripts share a common amino acid sequence until residue560, featuring a common signal peptide and six extracellular Ig-likedomains. FcRH5a represents a 759 amino acid secreted glycoprotein witheight Ig-like domains followed by 13 unique, predominantly polar aminoacids at its C-terminus. FcRH5b diverges from FcRH5a at amino acidresidue 560 and extends for a short stretch of 32 additional residues,whose hydrophobicity is compatible with its docking to the plasmamembranevia a GPI anchor. FcRH5c is the longest isoform whose sequencedeviates from FcRH5a at amino acid 746. FcRH5c encodes a 977 aa type Itransmembrane glycoprotein with nine extracellular Ig-type domains,harboring eight potential N-linked glycosylation sites, a 23 amino acidtransmembrane, and a 104 amino acid cytoplasmic domain with threeconsensus SH2 binding motifs with the ITIM consensus.

The FcRH genes are clustered together in the midst of the classical FcRgenes, FcγRI, FcγRII, FcγRIII, and FcεRI, in the 1q21-23 region ofchromosome 1. This region contains 1 of the most frequent secondarychromosomal abnormalities associated with malignant phenotype inhematopoietic tumors, especially in multiple myeloma (Hatzivassiliou G.et al. Immunity (2001) 14:277-89). FcRH5 is expressed only in the B-celllineage, starting as early as pre-B-cells, but does not attain fullexpression until the mature B-cell stage. Unlike most knownotherB-cell-specific surface proteins (e.g., CD20, CD19, and CD22), FcRH5continues to be expressed in plasma cells whereas other B-cell-specificmarkers are downregulated (Polson A G et al., Int Immunol (2006)18:1363-73). In addition, FcRH5 mRNA is overexpressed in multiplemyeloma cell lines with 1q21 abnormalities as detected byoligonucleotide arrays (Inoue J., Am J Pathol (2004) 165:71-81). Theexpression pattern indicates that FcRH5 could be a target forantibody-based therapies for the treatment of multiple myeloma. Multiplemyeloma is a malignancy of plasma cells characterized by skeletallesions, renal failure, anemia, and hypercalcemia. It is essentiallyincurable by current therapies. Current drug treatments for multiplemyeloma include combinations of the proteosome inhibitor bortezomib(Velcade), the immunomodulator lenalidomide (Revlimid), and the steroiddexamethasone.

FcRH5c specific antibody-based therapies and detection methods may beparticularly efficacious as they specifically recognize target cell,membrane-associated FcRH5 rather than antibodies which recognize bothsoluble and membrane isoforms of FcRH5. However, only the last Ig-likedomain of FcRH5 (Ig-like domain 9) is unique extracellular region thatdifferentiates between the three major isoforms of FcRH5, and there issignificant homology between the Ig-like domains within FcRH5. Further,the last Ig-like domain is highly conserved between FcRH1, FcRH2, FcRH3,and FcRH5. Any antibody-based therapy that specifically targeted FcRH5would have to have minimal cross-reactivity with other FcRHs to avoidadverse off-target effects (e.g., FcRH3 is expressed on normal NKcells). There is a need in the art for agents that aid in the diagnosisand treatment of cancer, such as FcRH5-associated cancer.

SUMMARY

Provided herein are anti-FcRH5 antibodies including bispecificantibodies, immunoconjugates, and methods of using the same. Providedherein are isolated anti-FcRH5 antibodies that binds an isoformc-specific region of the extracellular domain of FcRH5c. In someembodiments, the isoform c-specific region comprises Ig-like domain 9.In some embodiments, the isoform c-specific region comprises amino acids743-850 of SEQ ID NO:1.

In some embodiments, the antibody comprises: a) a heavy chain comprisinga HVR-H1 comprising the amino acid sequence of SEQ ID NO:38, HVR-H2comprising the amino acid sequence of SEQ ID NO:62, and HVR-H3comprising the amino acid sequence of SEQ ID NO:86; and/or b) a lightchain comprising a HVR-L1 comprising the amino acid sequence of SEQ IDNO:2, HVR-L2 comprising the amino acid sequence of SEQ ID NO:14, andHVR-L3 comprising the amino acid sequence of SEQ ID NO:26. In someembodiments, the heavy chain comprising a HVR-H1 comprising the aminoacid sequence of SEQ ID NO:50, HVR-H2 comprising the amino acid sequenceof SEQ ID NO:74, and HVR-H3 comprising the amino acid sequence of SEQ IDNO:98.

In some embodiments, the antibody comprises: a) a heavy chain comprisinga HVR-H1 comprising the amino acid sequence of SEQ ID NO:39, HVR-H2comprising the amino acid sequence of SEQ ID NO:63, and HVR-H3comprising the amino acid sequence of SEQ ID NO:87; and/or b) a lightchain comprising a HVR-L1 comprising the amino acid sequence of SEQ IDNO:3, HVR-L2 comprising the amino acid sequence of SEQ ID NO:15, andHVR-L3 comprising the amino acid sequence of SEQ ID NO:27. In someembodiments, the heavy chain comprising a HVR-H1 comprising the aminoacid sequence of SEQ ID NO:51, HVR-H2 comprising the amino acid sequenceof SEQ ID NO:75, and HVR-H3 comprising the amino acid sequence of SEQ IDNO:99.

In some embodiments, the antibody comprises: a) a heavy chain comprisinga HVR-H1 comprising the amino acid sequence of SEQ ID NO:40, HVR-H2comprising the amino acid sequence of SEQ ID NO:64, and HVR-H3comprising the amino acid sequence of SEQ ID NO:88; and/or b) a lightchain comprising a HVR-L1 comprising the amino acid sequence of SEQ IDNO:4, HVR-L2 comprising the amino acid sequence of SEQ ID NO:16, andHVR-L3 comprising the amino acid sequence of SEQ ID NO:28. In someembodiments, the heavy chain comprising a HVR-H1 comprising the aminoacid sequence of SEQ ID NO:52, HVR-H2 comprising the amino acid sequenceof SEQ ID NO:76, and HVR-H3 comprising the amino acid sequence of SEQ IDNO:100.

In some embodiments, the antibody comprises: a) a heavy chain comprisinga HVR-H1 comprising the amino acid sequence of SEQ ID NO:41, HVR-H2comprising the amino acid sequence of SEQ ID NO:65, and HVR-H3comprising the amino acid sequence of SEQ ID NO:89; and/or b) a lightchain comprising a HVR-L1 comprising the amino acid sequence of SEQ IDNO:5, HVR-L2 comprising the amino acid sequence of SEQ ID NO:17, andHVR-L3 comprising the amino acid sequence of SEQ ID NO:29. In someembodiments, the heavy chain comprising a HVR-H1 comprising the aminoacid sequence of SEQ ID NO:53, HVR-H2 comprising the amino acid sequenceof SEQ ID NO:77, and HVR-H3 comprising the amino acid sequence of SEQ IDNO:101.

In some embodiments, the antibody comprises: a) a heavy chain comprisinga HVR-H1 comprising the amino acid sequence of SEQ ID NO:42, HVR-H2comprising the amino acid sequence of SEQ ID NO:66, and HVR-H3comprising the amino acid sequence of SEQ ID NO:90; and/or b) a lightchain comprising a HVR-L1 comprising the amino acid sequence of SEQ IDNO:6, HVR-L2 comprising the amino acid sequence of SEQ ID NO:18, andHVR-L3 comprising the amino acid sequence of SEQ ID NO:30. In someembodiments, the heavy chain comprising a HVR-H1 comprising the aminoacid sequence of SEQ ID NO:54, HVR-H2 comprising the amino acid sequenceof SEQ ID NO:78, and HVR-H3 comprising the amino acid sequence of SEQ IDNO:102.

In some embodiments, the antibody comprises: a) a heavy chain comprisinga HVR-H1 comprising the amino acid sequence of SEQ ID NO:43, HVR-H2comprising the amino acid sequence of SEQ ID NO:67, and HVR-H3comprising the amino acid sequence of SEQ ID NO:91; and/or b) a lightchain comprising a HVR-L1 comprising the amino acid sequence of SEQ IDNO:7, HVR-L2 comprising the amino acid sequence of SEQ ID NO:19, andHVR-L3 comprising the amino acid sequence of SEQ ID NO:31. In someembodiments, the heavy chain comprising a HVR-H1 comprising the aminoacid sequence of SEQ ID NO:55, HVR-H2 comprising the amino acid sequenceof SEQ ID NO:79, and HVR-H3 comprising the amino acid sequence of SEQ IDNO:103.

In some embodiments, the antibody comprises: a) a heavy chain comprisinga HVR-H1 comprising the amino acid sequence of SEQ ID NO:44, HVR-H2comprising the amino acid sequence of SEQ ID NO:68, and HVR-H3comprising the amino acid sequence of SEQ ID NO:92; and/or b) a lightchain comprising a HVR-L1 comprising the amino acid sequence of SEQ IDNO:8, HVR-L2 comprising the amino acid sequence of SEQ ID NO:20, andHVR-L3 comprising the amino acid sequence of SEQ ID NO:32. In someembodiments, the heavy chain comprising a HVR-H1 comprising the aminoacid sequence of SEQ ID NO:56, HVR-H2 comprising the amino acid sequenceof SEQ ID NO:80, and HVR-H3 comprising the amino acid sequence of SEQ IDNO:104.

In some embodiments, the antibody comprises: a) a heavy chain comprisinga HVR-H1 comprising the amino acid sequence of SEQ ID NO:45, HVR-H2comprising the amino acid sequence of SEQ ID NO:69, and HVR-H3comprising the amino acid sequence of SEQ ID NO:93; and/or b) a lightchain comprising a HVR-L1 comprising the amino acid sequence of SEQ IDNO:9, HVR-L2 comprising the amino acid sequence of SEQ ID NO:21, andHVR-L3 comprising the amino acid sequence of SEQ ID NO:33. In someembodiments, the heavy chain comprising a HVR-H1 comprising the aminoacid sequence of SEQ ID NO:57, HVR-H2 comprising the amino acid sequenceof SEQ ID NO:81, and HVR-H3 comprising the amino acid sequence of SEQ IDNO:105.

In some embodiments, the antibody comprises: a) a heavy chain comprisinga HVR-H1 comprising the amino acid sequence of SEQ ID NO:46, HVR-H2comprising the amino acid sequence of SEQ ID NO:70, and HVR-H3comprising the amino acid sequence of SEQ ID NO:94; and/or b) a lightchain comprising a HVR-L1 comprising the amino acid sequence of SEQ IDNO:10, HVR-L2 comprising the amino acid sequence of SEQ ID NO:22, andHVR-L3 comprising the amino acid sequence of SEQ ID NO:34. In someembodiments, the heavy chain comprising a HVR-H1 comprising the aminoacid sequence of SEQ ID NO:58, HVR-H2 comprising the amino acid sequenceof SEQ ID NO:82, and HVR-H3 comprising the amino acid sequence of SEQ IDNO:106.

In some embodiments, the antibody comprises: a) a heavy chain comprisinga HVR-H1 comprising the amino acid sequence of SEQ ID NO:47, HVR-H2comprising the amino acid sequence of SEQ ID NO:71, and HVR-H3comprising the amino acid sequence of SEQ ID NO:95; and/or b) a lightchain comprising a HVR-L1 comprising the amino acid sequence of SEQ IDNO:11, HVR-L2 comprising the amino acid sequence of SEQ ID NO:23, andHVR-L3 comprising the amino acid sequence of SEQ ID NO:35. In someembodiments, the heavy chain comprising a HVR-H1 comprising the aminoacid sequence of SEQ ID NO:59, HVR-H2 comprising the amino acid sequenceof SEQ ID NO:83, and HVR-H3 comprising the amino acid sequence of SEQ IDNO:107.

In some embodiments, the antibody comprises: a) a heavy chain comprisinga HVR-H1 comprising the amino acid sequence of SEQ ID NO:48, HVR-H2comprising the amino acid sequence of SEQ ID NO:72, and HVR-H3comprising the amino acid sequence of SEQ ID NO:96; and/or b) a lightchain comprising a HVR-L1 comprising the amino acid sequence of SEQ IDNO:12, HVR-L2 comprising the amino acid sequence of SEQ ID NO:24, andHVR-L3 comprising the amino acid sequence of SEQ ID NO:36. In someembodiments, the heavy chain comprising a HVR-H1 comprising the aminoacid sequence of SEQ ID NO:60, HVR-H2 comprising the amino acid sequenceof SEQ ID NO:84, and HVR-H3 comprising the amino acid sequence of SEQ IDNO:108.

In some embodiments, the antibody comprises: a) a heavy chain comprisinga HVR-H1 comprising the amino acid sequence of SEQ ID NO:49, HVR-H2comprising the amino acid sequence of SEQ ID NO:73, and HVR-H3comprising the amino acid sequence of SEQ ID NO:97; and/or b) a lightchain comprising a HVR-L1 comprising the amino acid sequence of SEQ IDNO:13, HVR-L2 comprising the amino acid sequence of SEQ ID NO:25, andHVR-L3 comprising the amino acid sequence of SEQ ID NO:37. In someembodiments, the heavy chain comprising a HVR-H1 comprising the aminoacid sequence of SEQ ID NO:61, HVR-H2 comprising the amino acid sequenceof SEQ ID NO:85, and HVR-H3 comprising the amino acid sequence of SEQ IDNO:109.

In some embodiments of any of the antibodies, the antibody comprises: a)a VH sequence having at least 95% sequence identity to the amino acidsequence of SEQ ID NO:111 and/or a VL sequence having at least 95%sequence identity to the amino acid sequence of SEQ ID NO: 110; b) a VHsequence having at least 95% sequence identity to the amino acidsequence of SEQ ID NO:113 and/or a VL sequence having at least 95%sequence identity to the amino acid sequence of SEQ ID NO:112; c) a VHsequence having at least 95% sequence identity to the amino acidsequence of SEQ ID NO:115 and/or a VL sequence having at least 95%sequence identity to the amino acid sequence of SEQ ID NO:114; d) a VHsequence having at least 95% sequence identity to the amino acidsequence of SEQ ID NO:117 and/or a VL sequence having at least 95%sequence identity to the amino acid sequence of SEQ ID NO:116; e) a VHsequence having at least 95% sequence identity to the amino acidsequence of SEQ ID NO:119 and/or a VL sequence having at least 95%sequence identity to the amino acid sequence of SEQ ID NO:118; f) a VHsequence having at least 95% sequence identity to the amino acidsequence of SEQ ID NO:121 and/or a VL sequence having at least 95%sequence identity to the amino acid sequence of SEQ ID NO:120; g) a VHsequence having at least 95% sequence identity to the amino acidsequence of SEQ ID NO:123 and/or a VL sequence having at least 95%sequence identity to the amino acid sequence of SEQ ID NO:122; h) a VHsequence having at least 95% sequence identity to the amino acidsequence of SEQ ID NO:125 and/or a VL sequence having at least 95%sequence identity to the amino acid sequence of SEQ ID NO:124; i) a VHsequence having at least 95% sequence identity to the amino acidsequence of SEQ ID NO:127 and/or a VL sequence having at least 95%sequence identity to the amino acid sequence of SEQ ID NO: 126; j) a VHsequence having at least 95% sequence identity to the amino acidsequence of SEQ ID NO:129 and/or a VL sequence having at least 95%sequence identity to the amino acid sequence of SEQ ID NO:128; k) a VHsequence having at least 95% sequence identity to the amino acidsequence of SEQ ID NO:131 and/or a VL sequence having at least 95%sequence identity to the amino acid sequence of SEQ ID NO:130; l) a VHsequence having at least 95% sequence identity to the amino acidsequence of SEQ ID NO:133 and/or a VL sequence having at least 95%sequence identity to the amino acid sequence of SEQ ID NO:132; or a VHsequence having at least 95% sequence identity to the amino acidsequence of SEQ ID NO:135 and/or a VL sequence having at least 95%sequence identity to the amino acid sequence of SEQ ID NO:134.

In some embodiments of any of the antibodies, the antibody comprises: a)a VH sequence of SEQ ID NO:111 and/or a VL sequence of SEQ ID NO:110; b)a VH sequence of SEQ ID NO:113 and/or a VL sequence of SEQ ID NO:112; c)a VH sequence of SEQ ID NO:115 and/or a VL sequence of SEQ ID NO:114; d)a VH sequence of SEQ ID NO:117 and/or a VL sequence of SEQ ID NO:116; e)a VH sequence of SEQ ID NO:119 and/or a VL sequence of SEQ ID NO:118; f)a VH sequence of SEQ ID NO:121 and/or a VL sequence of SEQ ID NO:120; g)a VH sequence of SEQ ID NO:123 and/or a VL sequence of SEQ ID NO:122; h)a VH sequence of SEQ ID NO:125 and/or a VL sequence of SEQ ID NO:124; i)a VH sequence of SEQ ID NO:127 and/or a VL sequence of SEQ ID NO:126; j)a VH sequence of SEQ ID NO:129 and/or a VL sequence of SEQ ID NO:128; k)a VH sequence of SEQ ID NO:131 and/or a VL sequence of SEQ ID NO:130; l)a VH sequence of SEQ ID NO:133 and/or a VL sequence of SEQ ID NO:132, orm) a VH sequence of SEQ ID NO:135 and/or a VL sequence of SEQ ID NO:134.

In some embodiments of any of the antibodies, the antibody is amonoclonal antibody. In some embodiments of any of the antibodies, theantibody is a human, humanized, or chimeric antibody. In someembodiments of any of the antibodies, the antibody is an antibodyfragment that binds FcRH5. In some embodiments of any of the antibodies,the antibody is an IgG1, IgG2a or IgG2b antibody.

In some embodiments of any of the antibodies, the antibody has one ormore of the following characteristics: a) cross reactive with fulllength human and cyno FcRH5, b) does not cross react with FcRH1, FcRH2,FcRH3, and/or FcRH4, c) binds to endogenous FcRH5, d) does not crossreact with FcRH5a, and e) does not cross react with another Ig-likedomain of FcRH5.

In some embodiments of any of the antibodies, the antibody is abispecific antibody. In some embodiments, the bispecific antibody bindsFcRH5 and CD3.

In some embodiments, an isolated nucleic acid that encodes an antibodydescribed herein is provided. In some embodiments, a host cellcomprising the nucleic acid is provided. In some embodiments, a methodof producing an antibody described herein is provided. In someembodiments, the method comprises culturing the host cell comprising thenucleic acid that encodes an antibody.

In some embodiments, immunoconjugates are provided. In some embodiments,an immunoconjugate comprises an anti-FcRH5 antibody and a cytotoxicagent. In some embodiments, the anti-FcRH5 antibody binds an isoformc-specific region of the extracellular domain of FcRH5c. In someembodiments, the anti-FcRH5 antibodies binds Ig-like domain 9 of FcRH5c.In some embodiments, an immunoconjugate has the formula Ab-(L-D)p,wherein: (a) Ab is an antibody described herein; (b) L is a linker; (c)D is a drug selected from a maytansinoid, an auristatin, acalicheamicin, a pyrrolobenzodiazepine, and a nemorubicin derivative;and (d) p ranges from 1-8. In some embodiments, D is an auristatin. Insome such embodiments, D has formula D_(E)

wherein R² and R⁶ are each methyl, R³ and R⁴ are each isopropyl, R⁵ isH, R⁷ is sec-butyl, each R⁸ is independently selected from CH₃, O—CH₃,OH, and H; R⁹ is H; and R¹⁸ is —C(R⁸)₂—C(R⁸)₂-aryl. In some embodiments,D is MMAE having the structure:

In some embodiments, D is a pyrrolobenzodiazepine of Formula A:

wherein the dotted lines indicate the optional presence of a double bondbetween C1 and C2 or C2 and C3; R² is independently selected from H, OH,═O, ═CH₂, CN, R, OR, ═CH—R¹, ═C(R^(D))₂, O—SO₂—R, CO₂R and COR, andoptionally further selected from halo or dihalo, wherein R^(D) isindependently selected from R, CO₂R, COR, CHO, CO₂H, and halo; R⁶ and R⁹are independently selected from H, R, OH, OR, SH, SR, NH₂, NHR, NRR′,NO₂, Me₃Sn and halo; R⁷ is independently selected from H, R, OH, OR, SH,SR, NH₂, NHR, NRR′, NO₂, Me₃Sn and halo; Q is independently selectedfrom O, S and NH; R¹¹ is either H, or R or, where Q is O, SO₃M, where Mis a metal cation; R and R′ are each independently selected fromoptionally substituted C₁₋₈ alkyl, C₁₋₁₂ alkyl, C₃₋₈ heterocyclyl, C₃₋₂₀heterocyclyl, and C₅₋₂₀ aryl groups, and optionally in relation to thegroup NRR′, R and R′ together with the nitrogen atom to which they areattached form an optionally substituted 4-, 5-, 6- or 7-memberedheterocyclic ring; R¹², R¹⁶, R¹⁹ and R¹⁷ are as defined for R², R⁶, R⁹and R⁷ respectively; R″ is a C₃₋₁₂ alkylene group, which chain may beinterrupted by one or more heteroatoms and/or aromatic rings that areoptionally substituted; and X and X′ are independently selected from O,S and N(H). In some such embodiments, D is

wherein n is 0 or 1.

In some embodiments, D is a nemorubicin derivative. In some embodiments,D has a structure selected from:

In some embodiments, an immunoconjugate comprises a linker that iscleavable by a protease. In some embodiments, the linker comprises aval-cit dipeptide or a Phe-homoLys dipeptide. In some embodiments, animmunoconjugate comprises a linker that is acid-labile. In some suchembodiments, the linker comprises hydrazone.

In some embodiments, an immunoconjugate has a formula selected from:

wherein S is a sulfur atom;

In some embodiments, p ranges from 2-5.

In some embodiments, pharmaceutical formulations are provided. In somesuch embodiments, a pharmaceutical formulation comprises animmunoconjugate comprising an antibody that binds FcRH5, e.g., asdescribed herein. In some embodiments, the anti-FcRH5 antibody binds anisoform c-specific region of the extracellular domain of FcRH5c. In someembodiments, the anti-FcRH5 antibodies binds Ig-like domain 9 of FcRH5c.In some embodiments, a pharmaceutical formulation further comprises anadditional therapeutic agent.

In some embodiments, methods of treating individuals having FcRH5 (e.g.,FcRH5c)-positive cancers are provided. In some such embodiments, amethod comprises administering a pharmaceutical formulation comprisingan immunoconjugate comprising an antibody that binds FcRH5 and/or anFcRH5 bispecific antibody, e.g., as described herein. In someembodiments, the FcRH5 bispecific antibody comprises an FcRH5 bindingarm and a CD3 binding arm. In some embodiments, the anti-FcRH5 antibodybinds an isoform c-specific region of the extracellular domain ofFcRH5c. In some embodiments, the anti-FcRH5 antibodies binds Ig-likedomain 9 of FcRH5c. In some embodiments, the FcRH5-positive cancer is aB-cell proliferative disorder. In some embodiments, the FcRH5-positivecancer is plasma cell neoplasm. In some embodiments, the plasma cellneoplasm is multiple myeloma. In some embodiments, a method comprisesadministering an additional therapeutic agent to the individual.

In some embodiments, methods of inhibiting proliferation of an FcRH5(e.g., FcRH5c)-positive cell are provided. In some embodiments, themethod comprising exposing the cell to an immunoconjugate comprising anantibody that binds FcRH5 and/or an FcRH5 bispecific antibody underconditions permissive for binding of the antibody to FcRH5 on thesurface of the cell. In some embodiments, the FcRH5 bispecific antibodycomprises an FcRH5 binding arm and a CD3 binding arm. In someembodiments, the anti-FcRH5 antibody binds an isoform c-specific regionof the extracellular domain of FcRH5c. In some embodiments, theanti-FcRH5 antibodies binds Ig-like domain 9 of FcRH5c. In someembodiments, the antibody that binds FcRH5 is an antibody describedherein. In some embodiments, the FcRH5-positive cancer is a B-cellproliferative disorder. In some embodiments, the FcRH5-positive canceris plasma cell neoplasm. In some embodiments, the plasma cell neoplasmis multiple myeloma. In some embodiments, a method comprisesadministering an additional therapeutic agent to the individual.

In some embodiments, an antibody that binds FcRH5 is conjugated to alabel. In some embodiments, the anti-FcRH5 antibody binds an isoformc-specific region of the extracellular domain of FcRH5c. In someembodiments, the anti-FcRH5 antibodies binds Ig-like domain 9 of FcRH5c.In some embodiments, the antibody that binds FcRH5 is an antibodydescribed herein. In some embodiments, the label is a positron emitter.In some embodiments, the positron emitter is ⁸⁹Zr.

In some embodiments, a method of detecting human FcRH5 in a biologicalsample is provided. In some embodiments, a method comprises contactingthe biological sample with an anti-FcRH5 antibody under conditionspermissive for binding of the anti-FcRH5 antibody to a naturallyoccurring human FcRH5, and detecting whether a complex is formed betweenthe anti-FcRH5 antibody and a naturally occurring human FcRH5 in thebiological sample. In some embodiments, the anti-FcRH5 antibody binds anisoform c-specific region of the extracellular domain of FcRH5c. In someembodiments, the anti-FcRH5 antibodies binds Ig-like domain 9 of FcRH5c.In some embodiments, the anti-FcRH5 antibody is an antibody describedherein.

In some embodiments, a method for detecting an FcRH5-positive cancer isprovided. In some such embodiments, a method comprises (i) administeringa labeled anti-FcRH5 antibody to a subject having or suspected of havingan FcRH5-positive cancer, and (ii) detecting the labeled anti-FcRH5antibody in the subject, wherein detection of the labeled anti-FcRH5antibody indicates an FcRH5-positive cancer in the subject. In someembodiments, the anti-FcRH5 antibody binds an isoform c-specific regionof the extracellular domain of FcRH5c. In some embodiments, theanti-FcRH5 antibodies binds Ig-like domain 9 of FcRH5c. In someembodiments, an anti-FcRH5 antibody is an antibody described herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1(A) depicts the three major isoforms of FcRH5, FcRH5a (IRTA2a;UniProt Identifier Q96RD9-3), FcRH5b (IRTA2b; UniProt IdentifierQ96RD9-4), and FcRH5c (IRTA2c; UniProt Identifier Q96RD9-1). The Ig-likedomains are numbered and correspond to the amino acid sequence ofUniProt Identifier Q69RD9-1 (SEQ ID NO:1): Ig-like domain 1 (aa (“aminoacid”) 23-100), Ig-like domain 2 (aa 105-185), Ig-like domain 3 (aa188-271), Ig-like domain 4 (287-373), Ig-like domain 5 (aa 380-466),Ig-like domain 6 (aa 490-555), Ig-like domain 7 (aa 568-652), Ig-likedomain 8 (aa 658-731), and Ig-like domain 9 (aa 754-835).

FIG. 1(B) depicts part of FcRH5 (SEQ ID NO:136) and the structure andhomology of FcRH5 amino acids 735 to 977 of FcRH5c (SEQ ID NO:2).

FIG. 2(A) shows binding of FcRH5 antibodies to SVT2 cells transfectedwith human FcRH5 in different concentrations.

FIG. 2(B) shows binding of FcRH5 antibodies to SVT2 cells transfectedwith cyno FcRH5 in different concentrations.

FIG. 3(A) shows binding of FcRH5 antibodies to EJM cells transfectedwith human FcRH5.

FIG. 3(B) shows binding of FcRH5 antibodies to OPM2 cells transfectedwith human FcRH5.

FIG. 3(C) shows binding of 5A10.1 subclone supernatants to MOLP2 cellswhich express FcRH5 endogenously.

FIG. 3(D) shows binding of 5F1.1 subclone supernatants to MOLP2 cellswhich express FcRH5 endogenously.

FIG. 3(E) shows binding of 3G7.1 subclone supernatants to MOLP2 cellswhich express FcRH5 endogenously.

FIG. 3(F) shows binding of 6D2.2 subclone supernatants to MOLP2 cellswhich express FcRH5 endogenously.

FIG. 4(A) shows binding of FcRH5 subclone supernatants to 293 cellstransfected with mutant FcRH5 with deletion of 4 membrane proximalextracellular domains.

FIG. 4(B) shows binding of FcRH5 subclone supernatants to 293 cellstransfected with WT.

FIG. 5(A) shows binding of the FcRH5 antibodies to FcRH5a by ELISA.

FIG. 5(B) shows binding of FcRH5 subclone supernatants to human B cells.

FIG. 6(A) shows binding of FcRH5 subclone supernatants to SVT2 cellstransfected with FcRH1.

FIG. 6(B) shows binding of FcRH5 subclone supernatants to SVT2 cellstransfected with FcRH2.

FIG. 6(C) shows binding of FcRH5 subclone supernatants to SVT2 cellstransfected with FcRH3.

FIG. 6(D) shows binding of FcRH5 subclone supernatants to SVT2 cellstransfected with FcRH4.

FIG. 7 shows the binding of FcRH5 antibody subclone supernatants to NKcells.

FIG. 8(A) shows killing activity of FcRH5 bisFabs, FcRH5-TDB (clone10A8) and HER2-TDB on FcRH5 transfected 293 cells.

FIG. 8(B) shows killing activity of additional FcRH5 bisFabs incomparison with FCRH5-TDB and HER2-TDB on FcRH5 transfected 293 cells.

FIG. 8(C) shows killing activity of FcRH5-bisFabs and FcRH5-TDBsincorporating 2H7 or 3G7 as target arms on FcRH5 transfected 293 cells.

FIG. 8(D) shows killing activity of FcRH5-bisFabs incorporating 2H7 astarget arms and FcRH5-TDBs incorporating 5A10 as target arms on FcRH5transfected 293 cells.

FIG. 9(A) shows killing activity of FcRH5 bisFabs and FcRH5-TDBs onMOLP-2 cells.

FIG. 9(B) shows T-cell activation of FcRH5 bisFabs and FcRH5-TDBs onMOLP-2 cells.

DETAILED DESCRIPTION I. Definitions

The term “FcRH5,” as used herein, refers to any native, mature FcRH5which results from processing of an FcRH5 precursor protein in a cell.The term includes FcRH5 from any vertebrate source, including mammalssuch as primates (e.g. humans and cynomolgus monkeys) and rodents (e.g.,mice and rats), unless otherwise indicated. The term also includesnaturally occurring variants of FcRH5, e.g., splice variants or allelicvariants. In some embodiments, the amino acid sequences human FcRH5proteins is FcRH5a (IRTA2a; UniProt Identifier Q96RD9-3; 759 aa), FcRH5b(IRTA2b; UniProt Identifier Q96RD9-4; 592 aa), FcRH5c (IRTA2c; UniProtIdentifier Q96RD9-1; 977 aa (SEQ ID NO:1), UniProt Identifier Q96RD9-2(124 aa), and/or FcRH5d (IRTA2d; UniProt Identifier Q96RD9-5; 152 aa).

The term “glycosylated forms of FcRH5” refers to naturally occurringforms of FcRH5 that are post-translationally modified by the addition ofcarbohydrate residues.

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For purposes herein, however, % amino acid sequence identity values aregenerated using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc., and the source code has been filed with user documentation in theU.S. Copyright Office, Washington D.C., 20559, where it is registeredunder U.S. Copyright Registration No. TXU510087. The ALIGN-2 program ispublicly available from Genentech, Inc., South San Francisco, Calif., ormay be compiled from the source code. The ALIGN-2 program should becompiled for use on a UNIX operating system, including digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y where X is the number of amino acid residuesscored as identical matches by the sequence alignment program ALIGN-2 inthat program's alignment of A and B, and where Y is the total number ofamino acid residues in B. It will be appreciated that where the lengthof amino acid sequence A is not equal to the length of amino acidsequence B, the % amino acid sequence identity of A to B will not equalthe % amino acid sequence identity of B to A. Unless specifically statedotherwise, all % amino acid sequence identity values used herein areobtained as described in the immediately preceding paragraph using theALIGN-2 computer program.

The terms “anti-FcRH5 antibody” and “an antibody that binds to FcRH5”refer to an antibody that is capable of binding FcRH5 with sufficientaffinity such that the antibody is useful as a diagnostic and/ortherapeutic agent in targeting FcRH5. In one embodiment, the extent ofbinding of an anti-FcRH5 antibody to an unrelated, non-FcRH5 protein isless than about 10% of the binding of the antibody to FcRH5 as measured,e.g., by a radioimmunoassay (RIA). In certain embodiments, an antibodythat binds to FcRH5 has a dissociation constant (Kd) of ≦1 μM, ≦100 nM,≦10 nM, ≦5 Nm, ≦4 nM, ≦3 nM, ≦2 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001nM (e.g., 10⁻⁸M or less, e.g. from 10⁻⁸M to 10⁻¹³M, e.g., from 10⁻⁹M to10⁻¹³ M). In certain embodiments, an anti-FcRH5 antibody binds to anepitope of FcRH5 that is conserved among FcRH5 from different species.In some embodiments, the anti-FcRH5 antibody binds an isoform c-specificregion of the extracellular domain of FcRH5c. In some embodiments, theanti-FcRH5 antibodies binds Ig-like domain 9 of FcRH5c.

The term “antibody” is used herein in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as they exhibitthe desired antigen-binding activity.

An “antibody fragment” refers to a molecule other than an intactantibody that comprises a portion of an intact antibody and that bindsthe antigen to which the intact antibody binds. Examples of antibodyfragments include but are not limited to Fv, Fab, Fab′, Fab′-SH,F(ab′)₂; diabodies; linear antibodies; single-chain antibody molecules(e.g., scFv); and multispecific antibodies formed from antibodyfragments.

The term “epitope” refers to the particular site on an antigen moleculeto which an antibody binds.

An “antibody that binds to the same epitope” as a reference antibodyrefers to an antibody that blocks binding of the reference antibody toits antigen in a competition assay by 50% or more, and conversely, thereference antibody blocks binding of the antibody to its antigen in acompetition assay by 50% or more. An exemplary competition assay isprovided herein.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variant antibodies,e.g., containing naturally occurring mutations or arising duringproduction of a monoclonal antibody preparation, such variants generallybeing present in minor amounts. In contrast to polyclonal antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody of amonoclonal antibody preparation is directed against a single determinanton an antigen. Thus, the modifier “monoclonal” indicates the characterof the antibody as being obtained from a substantially homogeneouspopulation of antibodies, and is not to be construed as requiringproduction of the antibody by any particular method. For example, themonoclonal antibodies to be used in accordance with the presentinvention may be made by a variety of techniques, including but notlimited to the hybridoma method, recombinant DNA methods, phage-displaymethods, and methods utilizing transgenic animals containing all or partof the human immunoglobulin loci, such methods and other exemplarymethods for making monoclonal antibodies being described herein.

The terms “full length antibody,” “intact antibody,” and “wholeantibody” are used herein interchangeably to refer to an antibody havinga structure substantially similar to a native antibody structure orhaving heavy chains that contain an Fc region as defined herein.

A “naked antibody” refers to an antibody that is not conjugated to aheterologous moiety (e.g., a cytotoxic moiety) or radiolabel. The nakedantibody may be present in a pharmaceutical formulation.

“Native antibodies” refer to naturally occurring immunoglobulinmolecules with varying structures. For example, native IgG antibodiesare heterotetrameric glycoproteins of about 150,000 daltons, composed oftwo identical light chains and two identical heavy chains that aredisulfide-bonded. From N- to C-terminus, each heavy chain has a variableregion (VH), also called a variable heavy domain or a heavy chainvariable domain, followed by three constant domains (CH1, CH2, and CH3).Similarly, from N- to C-terminus, each light chain has a variable region(VL), also called a variable light domain or a light chain variabledomain, followed by a constant light (CL) domain. The light chain of anantibody may be assigned to one of two types, called kappa (κ) andlambda (λ), based on the amino acid sequence of its constant domain.

The term “chimeric” antibody refers to an antibody in which a portion ofthe heavy and/or light chain is derived from a particular source orspecies, while the remainder of the heavy and/or light chain is derivedfrom a different source or species.

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human or a human cellor derived from a non-human source that utilizes human antibodyrepertoires or other human antibody-encoding sequences. This definitionof a human antibody specifically excludes a humanized antibodycomprising non-human antigen-binding residues.

A “humanized” antibody refers to a chimeric antibody comprising aminoacid residues from non-human HVRs and amino acid residues from humanFRs. In certain embodiments, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the HVRs (e.g., CDRs) correspond tothose of a non-human antibody, and all or substantially all of the FRscorrespond to those of a human antibody. A humanized antibody optionallymay comprise at least a portion of an antibody constant region derivedfrom a human antibody. A “humanized form” of an antibody, e.g., anon-human antibody, refers to an antibody that has undergonehumanization.

The “class” of an antibody refers to the type of constant domain orconstant region possessed by its heavy chain. There are five majorclasses of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of thesemay be further divided into subclasses (isotypes), e.g., IgG₁, IgG₂,IgG₃, IgG₄, IgA₁, and IgA₂. The heavy chain constant domains thatcorrespond to the different classes of immunoglobulins are called α, δ,ε, γ, and μ, respectively.

The term “Fc region” herein is used to define a C-terminal region of animmunoglobulin heavy chain that contains at least a portion of theconstant region. The term includes native sequence Fc regions andvariant Fc regions. In one embodiment, a human IgG heavy chain Fc regionextends from Cys226, or from Pro230, to the carboxyl-terminus of theheavy chain. However, the C-terminal lysine (Lys447) of the Fc regionmay or may not be present. Unless otherwise specified herein, numberingof amino acid residues in the Fc region or constant region is accordingto the EU numbering system, also called the EU index, as described inKabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.,1991.

The term “variable region” or “variable domain” refers to the domain ofan antibody heavy or light chain that is involved in binding theantibody to antigen. The variable domains of the heavy chain and lightchain (VH and VL, respectively) of a native antibody generally havesimilar structures, with each domain comprising four conserved frameworkregions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindtet al. Kuby Immunology, 6^(th) ed., W.H. Freeman and Co., page 91(2007).) A single VH or VL domain may be sufficient to conferantigen-binding specificity. Furthermore, antibodies that bind aparticular antigen may be isolated using a VH or VL domain from anantibody that binds the antigen to screen a library of complementary VLor VH domains, respectively. See, e.g., Portolano et al., J. Immunol.150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

“Framework” or “FR” refers to variable domain residues other thanhypervariable region (HVR) residues. The FR of a variable domaingenerally consists of four FR domains: FR1, FR2, FR3, and FR4.Accordingly, the HVR and FR sequences generally appear in the followingsequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

A “human consensus framework” is a framework which represents the mostcommonly occurring amino acid residues in a selection of humanimmunoglobulin VL or VH framework sequences. Generally, the selection ofhuman immunoglobulin VL or VH sequences is from a subgroup of variabledomain sequences. Generally, the subgroup of sequences is a subgroup asin Kabat et al., Sequences of Proteins of Immunological Interest, FifthEdition, NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3. In oneembodiment, for the VL, the subgroup is subgroup kappa I as in Kabat etal., supra. In one embodiment, for the VH, the subgroup is subgroup IIIas in Kabat et al., supra.

The term “hypervariable region” or “HVR,” as used herein, refers to eachof the regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops (“hypervariable loops”).Generally, native four-chain antibodies comprise six HVRs; three in theVH (H1, H2, H3), and three in the VL (L1, L2, L3). HVRs generallycomprise amino acid residues from the hypervariable loops and/or fromthe “complementarity determining regions” (CDRs), the latter being ofhighest sequence variability and/or involved in antigen recognition.Exemplary hypervariable loops occur at amino acid residues 26-32 (L1),50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3).(Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987).) Exemplary CDRs(CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) occur at amino acidresidues 24-34 of L1, 50-56 of L2, 89-97 of L3, 31-35B of H1, 50-65 ofH2, and 95-102 of H3. (Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991).) With the exception of CDR1in VH, CDRs generally comprise the amino acid residues that form thehypervariable loops. CDRs also comprise “specificity determiningresidues,” or “SDRs,” which are residues that contact antigen. SDRs arecontained within regions of the CDRs called abbreviated-CDRs, or a-CDRs.Exemplary a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, anda-CDR-H3) occur at amino acid residues 31-34 of L1, 50-55 of L2, 89-96of L3, 31-35B of H1, 50-58 of H2, and 95-102 of H3. (See Almagro andFransson, Front. Biosci. 13:1619-1633 (2008).) Unless otherwiseindicated, HVR residues and other residues in the variable domain (e.g.,FR residues) are numbered herein according to Kabat et al., supra.

An “acceptor human framework” for the purposes herein is a frameworkcomprising the amino acid sequence of a light chain variable domain (VL)framework or a heavy chain variable domain (VH) framework derived from ahuman immunoglobulin framework or a human consensus framework, asdefined below. An acceptor human framework “derived from” a humanimmunoglobulin framework or a human consensus framework may comprise thesame amino acid sequence thereof, or it may contain amino acid sequencechanges. In some embodiments, the number of amino acid changes are 10 orless, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less,3 or less, or 2 or less. In some embodiments, the VL acceptor humanframework is identical in sequence to the VL human immunoglobulinframework sequence or human consensus framework sequence.

“Affinity” refers to the strength of the sum total of noncovalentinteractions between a single binding site of a molecule (e.g., anantibody) and its binding partner (e.g., an antigen). Unless indicatedotherwise, as used herein, “binding affinity” refers to intrinsicbinding affinity which reflects a 1:1 interaction between members of abinding pair (e.g., antibody and antigen). The affinity of a molecule Xfor its partner Y can generally be represented by the dissociationconstant (Kd). Affinity can be measured by common methods known in theart, including those described herein. Specific illustrative andexemplary embodiments for measuring binding affinity are described inthe following.

An “affinity matured” antibody refers to an antibody with one or morealterations in one or more hypervariable regions (HVRs), compared to aparent antibody which does not possess such alterations, suchalterations resulting in an improvement in the affinity of the antibodyfor antigen.

An “immunoconjugate” is an antibody conjugated to one or moreheterologous molecule(s), including but not limited to a cytotoxicagent.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents a cellular function and/or causes cell death ordestruction. Cytotoxic agents include, but are not limited to,radioactive isotopes (e.g., At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, sm¹⁵³,Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu); chemotherapeuticagents or drugs (e.g., methotrexate, adriamicin, vinca alkaloids(vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycinC, chlorambucil, daunorubicin or other intercalating agents); growthinhibitory agents; enzymes and fragments thereof such as nucleolyticenzymes; antibiotics; toxins such as small molecule toxins orenzymatically active toxins of bacterial, fungal, plant or animalorigin, including fragments and/or variants thereof; and the variousantitumor or anticancer agents disclosed below.

“Effector functions” refer to those biological activities attributableto the Fc region of an antibody, which vary with the antibody isotype.Examples of antibody effector functions include: C1 q binding andcomplement dependent cytotoxicity (CDC); Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g. B-cell receptor); and B-cellactivation.

An “isolated antibody” is one which has been separated from a componentof its natural environment. In some embodiments, an antibody is purifiedto greater than 95% or 99% purity as determined by, for example,electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillaryelectrophoresis) or chromatographic (e.g., ion exchange or reverse phaseHPLC). For review of methods for assessment of antibody purity, see,e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

An “isolated nucleic acid” refers to a nucleic acid molecule that hasbeen separated from a component of its natural environment. An isolatednucleic acid includes a nucleic acid molecule contained in cells thatordinarily contain the nucleic acid molecule, but the nucleic acidmolecule is present extrachromosomally or at a chromosomal location thatis different from its natural chromosomal location.

“Isolated nucleic acid encoding an anti-FcRH5 antibody” refers to one ormore nucleic acid molecules encoding antibody heavy and light chains (orfragments thereof), including such nucleic acid molecule(s) in a singlevector or separate vectors, and such nucleic acid molecule(s) present atone or more locations in a host cell.

The term “vector,” as used herein, refers to a nucleic acid moleculecapable of propagating another nucleic acid to which it is linked. Theterm includes the vector as a self-replicating nucleic acid structure aswell as the vector incorporated into the genome of a host cell intowhich it has been introduced. Certain vectors are capable of directingthe expression of nucleic acids to which they are operatively linked.Such vectors are referred to herein as “expression vectors.”

The terms “host cell,” “host cell line,” and “host cell culture” areused interchangeably and refer to cells into which exogenous nucleicacid has been introduced, including the progeny of such cells. Hostcells include “transformants” and “transformed cells,” which include theprimary transformed cell and progeny derived therefrom without regard tothe number of passages. Progeny may not be completely identical innucleic acid content to a parent cell, but may contain mutations. Mutantprogeny that have the same function or biological activity as screenedor selected for in the originally transformed cell are included herein.

As used herein, “treatment” (and grammatical variations thereof such as“treat” or “treating”) refers to clinical intervention in an attempt toalter the natural course of the individual being treated, and can beperformed either for prophylaxis or during the course of clinicalpathology. Desirable effects of treatment include, but are not limitedto, preventing occurrence or recurrence of disease, alleviation ofsymptoms, diminishment of any direct or indirect pathologicalconsequences of the disease, preventing metastasis, decreasing the rateof disease progression, amelioration or palliation of the disease state,and remission or improved prognosis. In some embodiments, antibodiesprovided herein are used to delay development of a disease or to slowthe progression of a disease.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth/proliferation. Examples of cancer include, butare not limited to, carcinoma, lymphoma (e.g., Hodgkin's andnon-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia. Moreparticular examples of such cancers include squamous cell cancer,small-cell lung cancer, non-small cell lung cancer, adenocarcinoma ofthe lung, squamous carcinoma of the lung, cancer of the peritoneum,hepatocellular cancer, gastrointestinal cancer, pancreatic cancer,glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer,hepatoma, breast cancer, colon cancer, colorectal cancer, smallintestine cancer, endometrial or uterine carcinoma, salivary glandcarcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer,thyroid cancer, hepatic carcinoma, leukemia and otherlymphoproliferative disorders, and various types of head and neckcancer.

A “B-cell malignancy” herein includes non-Hodgkin's lymphoma (NHL),including low grade/follicular NHL, small lymphocytic (SL) NHL,intermediate grade/follicular NHL, intermediate grade diffuse NHL, highgrade immunoblastic NHL, high grade lymphoblastic NHL, high grade smallnon-cleaved cell NHL, bulky disease NHL, mantle cell lymphoma,AIDS-related lymphoma, and Waldenstrom's Macroglobulinemia,non-Hodgkin's lymphoma (NHL), lymphocyte predominant Hodgkin's disease(LPHD), small lymphocytic lymphoma (SLL), chronic lymphocytic leukemia(CLL), indolent NHL including relapsed indolent NHL andrituximab-refractory indolent NHL; leukemia, including acutelymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), Hairycell leukemia, chronic myeloblastic leukemia; mantle cell lymphoma; andother hematologic malignancies. Such malignancies may be treated withantibodies directed against B-cell surface markers, such as FcRH5 (e.g.,FcRH5c). Such diseases are contemplated herein to be treated by theadministration of an antibody directed against a B-cell surface marker,such as FcRH5 (e.g., FcRH5c), and includes the administration of anunconjugated (“naked”) antibody or an antibody conjugated to a cytotoxicagent as disclosed herein. Such diseases are also contemplated herein tobe treated by combination therapy including an anti-FcRH5 antibody(including FcRH5 bispecific antibody) or anti-FcRH5 antibody drugconjugate in combination with another antibody or antibody drugconjugate, another cytotoxic agent, radiation or other treatmentadministered simultaneously or in series.

The term “non-Hodgkin's lymphoma” or “NHL”, as used herein, refers to acancer of the lymphatic system other than Hodgkin's lymphomas. Hodgkin'slymphomas can generally be distinguished from non-Hodgkin's lymphomas bythe presence of Reed-Sternberg cells in Hodgkin's lymphomas and theabsence of said cells in non-Hodgkin's lymphomas. Examples ofnon-Hodgkin's lymphomas encompassed by the term as used herein includeany that would be identified as such by one skilled in the art (e.g., anoncologist or pathologist) in accordance with classification schemesknown in the art, such as the Revised European-American Lymphoma (REAL)scheme as described in Color Atlas of Clinical Hematology (3rd edition),A. Victor Hoffbrand and John E. Pettit (eds.) (Harcourt Publishers Ltd.,2000). See, in particular, the lists in FIGS. 11.57, 11.58 and 11.59.More specific examples include, but are not limited to, relapsed orrefractory NHL, front line low grade NHL, Stage III/IV NHL, chemotherapyresistant NHL, precursor B lymphoblastic leukemia and/or lymphoma, smalllymphocytic lymphoma, B-cell chronic lymphocytic leukemia and/orprolymphocytic leukemia and/or small lymphocytic lymphoma, B-cellprolymphocytic lymphoma, immunocytoma and/or lymphoplasmacytic lymphoma,lymphoplasmacytic lymphoma, marginal zone B-cell lymphoma, splenicmarginal zone lymphoma, extranodal marginal zone—MALT lymphoma, nodalmarginal zone lymphoma, hairy cell leukemia, plasmacytoma and/or plasmacell myeloma, low grade/follicular lymphoma, intermediategrade/follicular NHL, mantle cell lymphoma, follicle center lymphoma(follicular), intermediate grade diffuse NHL, diffuse large B-celllymphoma, aggressive NHL (including aggressive front-line NHL andaggressive relapsed NHL), NHL relapsing after or refractory toautologous stem cell transplantation, primary mediastinal large B-celllymphoma, primary effusion lymphoma, high grade immunoblastic NHL, highgrade lymphoblastic NHL, high grade small non-cleaved cell NHL, bulkydisease NHL, Burkitt's lymphoma, precursor (peripheral) large granularlymphocytic leukemia, mycosis fungoides and/or Sezary syndrome, skin(cutaneous) lymphomas, anaplastic large cell lymphoma, angiocentriclymphoma.

Plasma cells disorders result from the uncontrolled division ormultiplication of a plasma cell clone. Plasma cells arise from activatedB lymphocytes (i.e., B-cells). Each B-cell produces a unique receptor,known as the B-cell receptor, arrayed on its cell surface that isspecific for a foreign substance, i.e., antigen. When a B-cell receptorbinds its cognate antigen, the cell expressing the receptor is activatedto re-enter the cell cycle, producing many clonal copies of itself. Theclones mature into plasma cells that reside principally in the bonemarrow and that are specialized to produce copies of the B-cell receptorthat are released into the blood stream as antibodies. In a plasma celldisorder, the plasma cell or the parent B-cell suffers genetic damageresulting in suppression of or insensitivity to the normal restraints oncell division and/or activity. Daughter plasma cells derived from suchcells are malignant in that they may divide unchecked and/or generateexcess amount of the same immunoglobulin (antibody). Often theimmunoglobulin produced is incomplete or has an incorrect conformationthat can result in accumulation of the protein (also known as monoclonalprotein, M protein, paraprotein or amyloid protein, dependent on thespecific disorder) in the serum, tissues or organs (especially thekidneys), leading to organ dysfunction and/or failure. Plasma celldisorders include monoclonal gammopathies of undetermined significance(MGUS), multiple myeloma (MM), macroglobulinemia, heavy chain diseases,and systemic light-chain amyloidosis (AL), which are differentiatedbased on the proliferative nature of the clone, the extent of marrowinvolvement, and the type of M protein expressed. Additional plasma celldisorders are solitary plasmacytoma, extramedullary plasmacytoma,multiple solitary plasmacytomas, plasma cell leukemia, Waldenstrom'smacroglobulinaemia, B-cell non-Hodgkin lymphomas, B-cell chroniclymphocytic leukemia.

The term “FcRH5-positive cancer” refers to a cancer comprising cellsthat express FcRH5 on their surface. For the purposes of determiningwhether a cell expresses FcRH5 on the surface, FcRH5 mRNA expression isconsidered to correlate to FcRH5 expression on the cell surface. In someembodiments, expression of FcRH5 mRNA is determined by a method selectedfrom in situ hybridization and RT-PCR (including quantitative RT-PCR).Alternatively, expression of FcRH5 on the cell surface can bedetermined, for example, using antibodies to FcRH5 in a method such asimmunohistochemistry, FACS, etc. In some embodiments, FcRH5 is one ormore of FcRH5a, FcRH5b, FcRH5c, UniProt Identifier Q96RD9-2, and/orFcRH5d. In some embodiments, the FcRH5 is FcRH5c.

The term “FcRH5-positive cell” refers to a cell that expresses FcRH5 onits surface. In some embodiments, FcRH5 is one or more of FcRH5a,FcRH5b, FcRH5c, UniProt Identifier Q96RD9-2, and/or FcRH5d. In someembodiments, the FcRH5 is FcRH5c.

An “effective amount” of an agent, e.g., a pharmaceutical formulation,refers to an amount effective, at dosages and for periods of timenecessary, to achieve the desired therapeutic or prophylactic result.

An “individual” or “subject” is a mammal. Mammals include, but are notlimited to, domesticated animals (e.g., cows, sheep, cats, dogs, andhorses), primates (e.g., humans and non-human primates such as monkeys),rabbits, and rodents (e.g., mice and rats). In certain embodiments, theindividual or subject is a human.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,combination therapy, contraindications and/or warnings concerning theuse of such therapeutic products.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of an activeingredient contained therein to be effective, and which contains noadditional components which are unacceptably toxic to a subject to whichthe formulation would be administered.

A “pharmaceutically acceptable carrier” refers to an ingredient in apharmaceutical formulation, other than an active ingredient, which isnontoxic to a subject. A pharmaceutically acceptable carrier includes,but is not limited to, a buffer, excipient, stabilizer, or preservative.

“Alkyl” is C₁-C₁₈ hydrocarbon containing normal, secondary, tertiary orcyclic carbon atoms. Examples are methyl (Me, —CH₃), ethyl (Et,—CH₂CH₃), 1-propyl (n-Pr, n-propyl, —CH₂CH₂CH₃), 2-propyl (i-Pr,i-propyl, —CH(CH₃)₂), 1-butyl (n-Bu, n-butyl, —CH₂CH₂CH₂CH₃),2-methyl-1-propyl (i-Bu, i-butyl, —CH₂CH(CH₃)₂), 2-butyl (s-Bu, s-butyl,—CH(CH₃)CH₂CH₃), 2-methyl-2-propyl (t-Bu, t-butyl, —C(CH₃)₃), 1-pentyl(n-pentyl, —CH₂CH₂CH₂CH₂CH₃), 2-pentyl (—CH(CH₃)CH₂CH₂CH₃), 3-pentyl(—CH(CH₂CH₃)₂), 2-methyl-2-butyl (—C(CH₃)₂CH₂CH₃), 3-methyl-2-butyl(—CH(CH₃)CH(CH₃)₂), 3-methyl-1-butyl (—CH₂CH₂CH(CH₃)₂), 2-methyl-1-butyl(—CH₂CH(CH₃)CH₂CH₃), 1-hexyl (—CH₂CH₂CH₂CH₂CH₂CH₃), 2-hexyl(—CH(CH₃)CH₂CH₂CH₂CH₃), 3-hexyl (—CH(CH₂CH₃)(CH₂CH₂CH₃)),2-methyl-2-pentyl (—C(CH₃)₂CH₂CH₂CH₃), 3-methyl-2-pentyl(—CH(CH₃)CH(CH₃)CH₂CH₃), 4-methyl-2-pentyl (—CH(CH₃)CH₂CH(CH₃)₂),3-methyl-3-pentyl (—C(CH₃)(CH₂CH₃)₂), 2-methyl-3-pentyl(—CH(CH₂CH₃)CH(CH₃)₂), 2,3-dimethyl-2-butyl (—C(CH₃)₂CH(CH₃)₂),3,3-dimethyl-2-butyl (—CH(CH₃)C(CH₃)₃. The term “C₁-C₈ alkyl,” as usedherein refers to a straight chain or branched, saturated or unsaturatedhydrocarbon having from 1 to 8 carbon atoms. Representative “C₁-C₈alkyl” groups include, but are not limited to, -methyl, -ethyl,-n-propyl, -n-butyl, -n-pentyl, -n-hexyl, -n-heptyl, -n-octyl, -n-nonyland -n-decyl; while branched C₁-C₈ alkyls include, but are not limitedto, -isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl,2-methylbutyl, unsaturated C₁-C₈ alkyls include, but are not limited to,-vinyl, -allyl, -1-butenyl, -2-butenyl, -isobutylenyl, -1-pentenyl,-2-pentenyl, -3-methyl-1-butenyl, -2-methyl-2-butenyl,-2,3-dimethyl-2-butenyl, 1-hexyl, 2-hexyl, 3-hexyl, -acetylenyl,-propynyl, -1-butynyl, -2-butynyl, -1-pentynyl, -2-pentynyl, -3-methyl-1butynyl. A C₁-C₈ alkyl group can be unsubstituted or substituted withone or more groups including, but not limited to, —C₁-C₈ alkyl,—O—(C₁-C₈ alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂,—C(O)NHR′, —C(O)N(R′)₂—NHC(O)R′, —SO₃R′, —S(O)₂R′, —S(O)R′, —OH,-halogen, —N₃, —NH₂, —NH(R′), —N(R′)₂ and —CN; where each R′ isindependently selected from H, —C₁-C₈ alkyl and aryl.

The term “C₁-C₁₂ alkyl,” as used herein refers to a straight chain orbranched, saturated or unsaturated hydrocarbon having from 1 to 12carbon atoms. A C₁-C₁₂ alkyl group can be unsubstituted or substitutedwith one or more groups including, but not limited to, —C₁-C₈ alkyl,—O—(C₁-C₈ alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂,—C(O)NHR′, —C(O)N(R′)₂—NHC(O)R′, —SO₃R′, —S(O)₂R′, —S(O)R′, —OH,-halogen, —N₃, —NH₂, —NH(R′), —N(R′)₂ and —CN; where each R′ isindependently selected from H, —C₁-C₈ alkyl and aryl.

The term “C₁-C₆ alkyl,” as used herein refers to a straight chain orbranched, saturated or unsaturated hydrocarbon having from 1 to 6 carbonatoms. Representative “C₁-C₆ alkyl” groups include, but are not limitedto, -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, -and n-hexyl; whilebranched C₁-C₆ alkyls include, but are not limited to, -isopropyl,-sec-butyl, -isobutyl, -tert-butyl, -isopentyl, and 2-methylbutyl;unsaturated C₁-C₆ alkyls include, but are not limited to, -vinyl,-allyl, -1-butenyl, -2-butenyl, and -isobutylenyl, -1-pentenyl,-2-pentenyl, -3-methyl-1-butenyl, -2-methyl-2-butenyl,-2,3-dimethyl-2-butenyl, 1-hexyl, 2-hexyl, and 3-hexyl. A C₁-C₆ alkylgroup can be unsubstituted or substituted with one or more groups, asdescribed above for C₁-C₈ alkyl group.

The term “C₁-C₄ alkyl,” as used herein refers to a straight chain orbranched, saturated or unsaturated hydrocarbon having from 1 to 4 carbonatoms. Representative “C₁-C₄ alkyl” groups include, but are not limitedto, -methyl, -ethyl, -n-propyl, -n-butyl; while branched C₁-C₄ alkylsinclude, but are not limited to, -isopropyl, -sec-butyl, -isobutyl,-tert-butyl; unsaturated C₁-C₄ alkyls include, but are not limited to,-vinyl, -allyl, -1-butenyl, -2-butenyl, and -isobutylenyl. A C₁-C₄ alkylgroup can be unsubstituted or substituted with one or more groups, asdescribed above for C₁-C₈ alkyl group.

“Alkoxy” is an alkyl group singly bonded to an oxygen. Exemplary alkoxygroups include, but are not limited to, methoxy (—OCH₃) and ethoxy(—OCH₂CH₃). A “C₁-C₅ alkoxy” is an alkoxy group with 1 to 5 carbonatoms. Alkoxy groups may can be unsubstituted or substituted with one ormore groups, as described above for alkyl groups.

“Alkenyl” is C₂-C₁₈ hydrocarbon containing normal, secondary, tertiaryor cyclic carbon atoms with at least one site of unsaturation, i.e. acarbon-carbon, sp² double bond. Examples include, but are not limitedto: ethylene or vinyl (—CH═CH₂), allyl (—CH₂CH═CH₂), cyclopentenyl(—C₅H₇), and 5-hexenyl (—CH₂CH₂CH₂CH₂CH═CH₂). A “C₂-C₈ alkenyl” is ahydrocarbon containing 2 to 8 normal, secondary, tertiary or cycliccarbon atoms with at least one site of unsaturation, i.e. acarbon-carbon, sp² double bond.

“Alkynyl” is C2-C18 hydrocarbon containing normal, secondary, tertiaryor cyclic carbon atoms with at least one site of unsaturation, i.e. acarbon-carbon, sp triple bond. Examples include, but are not limited to:acetylenic (—C≡CH) and propargyl (—CH₂C≡CH). A “C₂-C₈ alkynyl” is ahydrocarbon containing 2 to 8 normal, secondary, tertiary or cycliccarbon atoms with at least one site of unsaturation, i.e. acarbon-carbon, sp triple bond.

“Alkylene” refers to a saturated, branched or straight chain or cyclichydrocarbon radical of 1-18 carbon atoms, and having two monovalentradical centers derived by the removal of two hydrogen atoms from thesame or two different carbon atoms of a parent alkane. Typical alkyleneradicals include, but are not limited to: methylene (—CH₂—) 1,2-ethyl(—CH₂CH₂—), 1,3-propyl (—CH₂CH₂CH₂—), 1,4-butyl (—CH₂CH₂CH₂CH₂—), andthe like.

A “C₁-C₁₀ alkylene” is a straight chain, saturated hydrocarbon group ofthe formula —(CH₂)₁₋₁₀—. Examples of a C₁-C₁₀ alkylene includemethylene, ethylene, propylene, butylene, pentylene, hexylene,heptylene, ocytylene, nonylene and decalene.

“Alkenylene” refers to an unsaturated, branched or straight chain orcyclic hydrocarbon radical of 2-18 carbon atoms, and having twomonovalent radical centers derived by the removal of two hydrogen atomsfrom the same or two different carbon atoms of a parent alkene. Typicalalkenylene radicals include, but are not limited to: 1,2-ethylene(—CH═CH—).

“Alkynylene” refers to an unsaturated, branched or straight chain orcyclic hydrocarbon radical of 2-18 carbon atoms, and having twomonovalent radical centers derived by the removal of two hydrogen atomsfrom the same or two different carbon atoms of a parent alkyne. Typicalalkynylene radicals include, but are not limited to: acetylene (—C≡C—),propargyl (—CH₂C≡C—), and 4-pentynyl (—CH₂CH₂CH₂C≡C—).

“Aryl” refers to a carbocyclic aromatic group. Examples of aryl groupsinclude, but are not limited to, phenyl, naphthyl and anthracenyl. Acarbocyclic aromatic group or a heterocyclic aromatic group can beunsubstituted or substituted with one or more groups including, but notlimited to, —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -aryl, —C(O)R′, —OC(O)R′,—C(O)OR′, —C(O)NH₂, —C(O)NHR′, —C(O)N(R′)₂—NHC(O)R′, —S(O)₂R′, —S(O)R′,—OH, -halogen, —N₃, —NH₂, —NH(R′), —N(R′)₂ and —CN; wherein each R′ isindependently selected from H, —C₁-C₈ alkyl and aryl.

A “C₅-C₂₀ aryl” is an aryl group with 5 to 20 carbon atoms in thecarbocyclic aromatic rings. Examples of C₅-C₂₀ aryl groups include, butare not limited to, phenyl, naphthyl and anthracenyl. A C₅-C₂₀ arylgroup can be substituted or unsubstituted as described above for arylgroups. A “C₅-C₁₄ aryl” is an aryl group with 5 to 14 carbon atoms inthe carbocyclic aromatic rings. Examples of C₅-C₁₄ aryl groups include,but are not limited to, phenyl, naphthyl and anthracenyl. A C₅-C₁₄ arylgroup can be substituted or unsubstituted as described above for arylgroups.

An “arylene” is an aryl group which has two covalent bonds and can be inthe ortho, meta, or para configurations as shown in the followingstructures:

in which the phenyl group can be unsubstituted or substituted with up tofour groups including, but not limited to, —C₁-C₈ alkyl, —O—(C₁-C₈alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂, —C(O)NHR′,—C(O)N(R′)₂—NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH, -halogen, —N₃, —NH₂,—NH(R′), —N(R′)₂ and —CN; wherein each R′ is independently selected fromH, —C₁-C₈ alkyl and aryl.

“Arylalkyl” refers to an acyclic alkyl radical in which one of thehydrogen atoms bonded to a carbon atom, typically a terminal or sp³carbon atom, is replaced with an aryl radical. Typical arylalkyl groupsinclude, but are not limited to, benzyl, 2-phenylethan-1-yl,2-phenylethen-1-yl, naphthylmethyl, 2-naphthylethan-1-yl,2-naphthylethen-1-yl, naphthobenzyl, 2-naphthophenylethan-1-yl and thelike. The arylalkyl group comprises 6 to 20 carbon atoms, e.g. the alkylmoiety, including alkanyl, alkenyl or alkynyl groups, of the arylalkylgroup is 1 to 6 carbon atoms and the aryl moiety is 5 to 14 carbonatoms.

“Heteroarylalkyl” refers to an acyclic alkyl radical in which one of thehydrogen atoms bonded to a carbon atom, typically a terminal or sp³carbon atom, is replaced with a heteroaryl radical. Typicalheteroarylalkyl groups include, but are not limited to,2-benzimidazolylmethyl, 2-furylethyl, and the like. The heteroarylalkylgroup comprises 6 to 20 carbon atoms, e.g. the alkyl moiety, includingalkanyl, alkenyl or alkynyl groups, of the heteroarylalkyl group is 1 to6 carbon atoms and the heteroaryl moiety is 5 to 14 carbon atoms and 1to 3 heteroatoms selected from N, O, P, and S. The heteroaryl moiety ofthe heteroarylalkyl group may be a monocycle having 3 to 7 ring members(2 to 6 carbon atoms or a bicycle having 7 to 10 ring members (4 to 9carbon atoms and 1 to 3 heteroatoms selected from N, O, P, and S), forexample: a bicyclo[4,5], [5,5], [5,6], or [6,6] system.

“Substituted alkyl,” “substituted aryl,” and “substituted arylalkyl”mean alkyl, aryl, and arylalkyl respectively, in which one or morehydrogen atoms are each independently replaced with a substituent.

Typical substituents include, but are not limited to, —X, —R, —OR, —SR,—NR₂, —NR₃, ═NR, —CX₃, —CN, —OCN, —SCN, —N═C═O, —NCS, —NO, —NO₂, ═N₂,—N₃, NC(═O)R, —C(═O)R, —C(═O)NR₂, —SO₃H, —S(═O)₂R, —OS(═O)₂OR,—S(═O)₂NR, —S(═O)R, —OP(═O)(OR)₂, —P(═O)(OR)₂, —PO₃, —PO₃H₂, —C(═O)R,—C(═O)X, —C(═S)R, —CO₂R, —CO₂, —C(═S)OR, —C(═O)SR, —C(═S)SR, —C(═O)NR₂,—C(═S)NR₂, —C(═NR)NR₂, where each X is independently a halogen: F, Cl,Br, or I; and each R is independently —H, C₂-C₁₈ alkyl, C₆-C₂₀ aryl,C₃-C₁₄ heterocycle, protecting group or prodrug moiety. Alkylene,alkenylene, and alkynylene groups as described above may also besimilarly substituted.

“Heteroaryl” and “heterocycle” refer to a ring system in which one ormore ring atoms is a heteroatom, e.g. nitrogen, oxygen, and sulfur. Theheterocycle radical comprises 3 to 20 carbon atoms and 1 to 3heteroatoms selected from N, O, P, and S. A heterocycle may be amonocycle having 3 to 7 ring members (2 to 6 carbon atoms and 1 to 3heteroatoms selected from N, O, P, and S) or a bicycle having 7 to 10ring members (4 to 9 carbon atoms and 1 to 3 heteroatoms selected fromN, O, P, and S), for example: a bicyclo[4,5], [5,5], [5,6], or [6,6]system.

Exemplary heterocycles are described, e.g., in Paquette, Leo A.,“Principles of Modern Heterocyclic Chemistry” (W. A. Benjamin, New York,1968), particularly Chapters 1, 3, 4, 6, 7, and 9; “The Chemistry ofHeterocyclic Compounds, A series of Monographs” (John Wiley & Sons, NewYork, 1950 to present), in particular Volumes 13, 14, 16, 19, and 28;and J. Am. Chem. Soc. (1960) 82:5566.

Examples of heterocycles include by way of example and not limitationpyridyl, dihydroypyridyl, tetrahydropyridyl (piperidyl), thiazolyl,tetrahydrothiophenyl, sulfur oxidized tetrahydrothiophenyl, pyrimidinyl,furanyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, tetrazolyl,benzofuranyl, thianaphthalenyl, indolyl, indolenyl, quinolinyl,isoquinolinyl, benzimidazolyl, piperidinyl, 4-piperidonyl, pyrrolidinyl,2-pyrrolidonyl, pyrrolinyl, tetrahydrofuranyl, bis-tetrahydrofuranyl,tetrahydropyranyl, bis-tetrahydropyranyl, tetrahydroquinolinyl,tetrahydroisoquinolinyl, decahydroquinolinyl, octahydroisoquinolinyl,azocinyl, triazinyl, 6H-1,2,5-thiadiazinyl, 2H,6H-1,5,2-dithiazinyl,thienyl, thianthrenyl, pyranyl, isobenzofuranyl, chromenyl, xanthenyl,phenoxathinyl, 2H-pyrrolyl, isothiazolyl, isoxazolyl, pyrazinyl,pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, 1H-indazolyl, purinyl,4H-quinolizinyl, phthalazinyl, naphthyridinyl, quinoxalinyl,quinazolinyl, cinnolinyl, pteridinyl, 4aH-carbazolyl, carbazolyl,β-carbolinyl, phenanthridinyl, acridinyl, pyrimidinyl, phenanthrolinyl,phenazinyl, phenothiazinyl, furazanyl, phenoxazinyl, isochromanyl,chromanyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl,piperazinyl, indolinyl, isoindolinyl, quinuclidinyl, morpholinyl,oxazolidinyl, benzotriazolyl, benzisoxazolyl, oxindolyl, benzoxazolinyl,and isatinoyl.

By way of example and not limitation, carbon bonded heterocycles arebonded at position 2, 3, 4, 5, or 6 of a pyridine, position 3, 4, 5, or6 of a pyridazine, position 2, 4, 5, or 6 of a pyrimidine, position 2,3, 5, or 6 of a pyrazine, position 2, 3, 4, or 5 of a furan,tetrahydrofuran, thiofuran, thiophene, pyrrole or tetrahydropyrrole,position 2, 4, or 5 of an oxazole, imidazole or thiazole, position 3, 4,or 5 of an isoxazole, pyrazole, or isothiazole, position 2 or 3 of anaziridine, position 2, 3, or 4 of an azetidine, position 2, 3, 4, 5, 6,7, or 8 of a quinoline or position 1, 3, 4, 5, 6, 7, or 8 of anisoquinoline. Still more typically, carbon bonded heterocycles include2-pyridyl, 3-pyridyl, 4-pyridyl, 5-pyridyl, 6-pyridyl, 3-pyridazinyl,4-pyridazinyl, 5-pyridazinyl, 6-pyridazinyl, 2-pyrimidinyl,4-pyrimidinyl, 5-pyrimidinyl, 6-pyrimidinyl, 2-pyrazinyl, 3-pyrazinyl,5-pyrazinyl, 6-pyrazinyl, 2-thiazolyl, 4-thiazolyl, or 5-thiazolyl.

By way of example and not limitation, nitrogen bonded heterocycles arebonded at position 1 of an aziridine, azetidine, pyrrole, pyrrolidine,2-pyrroline, 3-pyrroline, imidazole, imidazolidine, 2-imidazoline,3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3-pyrazoline,piperidine, piperazine, indole, indoline, 1H-indazole, position 2 of aisoindole, or isoindoline, position 4 of a morpholine, and position 9 ofa carbazole, or β-carboline. Still more typically, nitrogen bondedheterocycles include 1-aziridyl, 1-azetedyl, 1-pyrrolyl, 1-imidazolyl,1-pyrazolyl, and 1-piperidinyl.

A “C₃-C₈ heterocycle” refers to an aromatic or non-aromatic C₃-C₈carbocycle in which one to four of the ring carbon atoms areindependently replaced with a heteroatom from the group consisting of O,S and N. Representative examples of a C₃-C₈ heterocycle include, but arenot limited to, benzofuranyl, benzothiophene, indolyl, benzopyrazolyl,coumarinyl, isoquinolinyl, pyrrolyl, thiophenyl, furanyl, thiazolyl,imidazolyl, pyrazolyl, triazolyl, quinolinyl, pyrimidinyl, pyridinyl,pyridonyl, pyrazinyl, pyridazinyl, isothiazolyl, isoxazolyl andtetrazolyl. A C₃-C₈ heterocycle can be unsubstituted or substituted withup to seven groups including, but not limited to, —C₁-C₈ alkyl,—O—(C₁-C₈ alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂,—C(O)NHR′, —C(O)N(R′)₂—NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH, -halogen, —N₃,—NH₂, —NH(R′), —N(R′)₂ and —CN; wherein each R′ is independentlyselected from H, —C₁-C₈ alkyl and aryl.

“C₃-C₈ heterocyclo” refers to a C₃-C₈ heterocycle group defined abovewherein one of the heterocycle group's hydrogen atoms is replaced with abond. A C₃-C₈ heterocyclo can be unsubstituted or substituted with up tosix groups including, but not limited to, —C₁-C₈ alkyl, —O—(C₁-C₈alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂, —C(O)NHR′,—C(O)N(R′)₂—NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH, -halogen, —N₃, —NH₂,—NH(R′), —N(R′)₂ and —CN; wherein each R′ is independently selected fromH, —C₁-C₈ alkyl and aryl.

A “C₃-C₂₀ heterocycle” refers to an aromatic or non-aromatic C₃-C₈carbocycle in which one to four of the ring carbon atoms areindependently replaced with a heteroatom from the group consisting of O,S and N. A C₃-C₂₀ heterocycle can be unsubstituted or substituted withup to seven groups including, but not limited to, —C₁-C₈ alkyl,—O—(C₁-C₈ alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂,—C(O)NHR′, —C(O)N(R′)₂—NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH, -halogen, —N₃,—NH₂, —NH(R′), —N(R′)₂ and —CN; wherein each R′ is independentlyselected from H, —C₁-C₈ alkyl and aryl.

“C₃-C₂₀ heterocyclo” refers to a C₃-C₂₀ heterocycle group defined abovewherein one of the heterocycle group's hydrogen atoms is replaced with abond.

“Carbocycle” means a saturated or unsaturated ring having 3 to 7 carbonatoms as a monocycle or 7 to 12 carbon atoms as a bicycle. Monocycliccarbocycles have 3 to 6 ring atoms, still more typically 5 or 6 ringatoms. Bicyclic carbocycles have 7 to 12 ring atoms, e.g. arranged as abicyclo[4,5], [5,5], [5,6] or [6,6] system, or 9 or 10 ring atomsarranged as a bicyclo[5,6] or [6,6] system. Examples of monocycliccarbocycles include cyclopropyl, cyclobutyl, cyclopentyl,1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl,1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl, cycloheptyl,and cyclooctyl.

A “C₃-C₈ carbocycle” is a 3-, 4-, 5-, 6-, 7- or 8-membered saturated orunsaturated non-aromatic carbocyclic ring. Representative C₃-C₈carbocycles include, but are not limited to, -cyclopropyl, -cyclobutyl,-cyclopentyl, -cyclopentadienyl, -cyclohexyl, -cyclohexenyl,-1,3-cyclohexadienyl, -1,4-cyclohexadienyl, -cycloheptyl,-1,3-cycloheptadienyl, -1,3,5-cycloheptatrienyl, -cyclooctyl, and-cyclooctadienyl. A C₃-C₈ carbocycle group can be unsubstituted orsubstituted with one or more groups including, but not limited to,—C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′,—C(O)NH₂, —C(O)NHR′, —C(O)N(R′)₂—NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH,-halogen, —N₃, —NH₂, —NH(R′), —N(R′)₂ and —CN; where each R′ isindependently selected from H, —C₁-C₈ alkyl and aryl.

A “C₃-C₈ carbocyclo” refers to a C₃-C₈ carbocycle group defined abovewherein one of the carbocycle groups' hydrogen atoms is replaced with abond.

“Linker” refers to a chemical moiety comprising a covalent bond or achain of atoms that covalently attaches an antibody to a drug moiety. Invarious embodiments, linkers include a divalent radical such as analkyldiyl, an aryldiyl, a heteroaryldiyl, moieties such as: —(CR₂)_(n)O(CR₂)_(n)—, repeating units of alkyloxy (e.g. polyethylenoxy, PEG,polymethyleneoxy) and alkylamino (e.g. polyethyleneamino, Jeffamine™);and diacid ester and amides including succinate, succinamide,diglycolate, malonate, and caproamide. In various embodiments, linkerscan comprise one or more amino acid residues, such as valine,phenylalanine, lysine, and homolysine.

The term “chiral” refers to molecules which have the property ofnon-superimposability of the mirror image partner, while the term“achiral” refers to molecules which are superimposable on their mirrorimage partner.

The term “stereoisomers” refers to compounds which have identicalchemical constitution, but differ with regard to the arrangement of theatoms or groups in space.

“Diastereomer” refers to a stereoisomer with two or more centers ofchirality and whose molecules are not mirror images of one another.Diastereomers have different physical properties, e.g. melting points,boiling points, spectral properties, and reactivities. Mixtures ofdiastereomers may separate under high resolution analytical proceduressuch as electrophoresis and chromatography.

“Enantiomers” refer to two stereoisomers of a compound which arenon-superimposable mirror images of one another.

Stereochemical definitions and conventions used herein generally followS. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984)McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S.,Stereochemistry of Organic Compounds (1994) John Wiley & Sons, Inc., NewYork. Many organic compounds exist in optically active forms, i.e., theyhave the ability to rotate the plane of plane-polarized light. Indescribing an optically active compound, the prefixes D and L, or R andS, are used to denote the absolute configuration of the molecule aboutits chiral center(s). The prefixes d and 1 or (+) and (−) are employedto designate the sign of rotation of plane-polarized light by thecompound, with (−) or 1 meaning that the compound is levorotatory. Acompound prefixed with (+) or d is dextrorotatory. For a given chemicalstructure, these stereoisomers are identical except that they are mirrorimages of one another. A specific stereoisomer may also be referred toas an enantiomer, and a mixture of such isomers is often called anenantiomeric mixture. A 50:50 mixture of enantiomers is referred to as aracemic mixture or a racemate, which may occur where there has been nostereoselection or stereospecificity in a chemical reaction or process.The terms “racemic mixture” and “racemate” refer to an equimolar mixtureof two enantiomeric species, devoid of optical activity.

“Leaving group” refers to a functional group that can be substituted byanother functional group. Certain leaving groups are well known in theart, and examples include, but are not limited to, a halide (e.g.,chloride, bromide, iodide), methanesulfonyl (mesyl), p-toluenesulfonyl(tosyl), trifluoromethylsulfonyl (triflate), andtrifluoromethylsulfonate.

The term “protecting group” refers to a substituent that is commonlyemployed to block or protect a particular functionality while reactingother functional groups on the compound. For example, an“amino-protecting group” is a substituent attached to an amino groupthat blocks or protects the amino functionality in the compound.Suitable amino-protecting groups include, but are not limited to,acetyl, trifluoroacetyl, t-butoxycarbonyl (BOC), benzyloxycarbonyl (CBZ)and 9-fluorenylmethylenoxycarbonyl (Fmoc). For a general description ofprotecting groups and their use, see T. W. Greene, Protective Groups inOrganic Synthesis, John Wiley & Sons, New York, 1991, or a lateredition.

As is understood by one skilled in the art, reference to “about” a valueor parameter herein includes (and describes) embodiments that aredirected to that value or parameter per se. For example, descriptionreferring to “about X” includes description of “X”.

It is understood that aspect and embodiments described herein include“consisting” and/or “consisting essentially of” aspects and embodiments.As used herein, the singular form “a”, “an”, and “the” includes pluralreferences unless indicated otherwise.

II. Compositions and Methods

Provided herein are antibodies that bind to FcRH5 including bispecificantibodies and immunoconjugates comprising such antibodies. Antibodiesand immunoconjugates may be useful, e.g., for the diagnosis or treatmentof FcRH5-positive cancers. In some embodiments, the anti-FcRH5 antibodybinds an isoform c-specific region of the extracellular domain ofFcRH5c. In some embodiments, the anti-FcRH5 antibodies bind Ig-likedomain 9 of FcRH5c.

Without being bound by theory, the selection of the precise antigen forthe antibodies of the present invention was driven by at least threeimportant considerations. First, there was a need for little to nocross-reactivity with FcRH5 isoforms other than FcRH5c, such as isoforma and isoform b, to avoid the resulting therapeutic from binding tonon-target molecules and thus reducing its effectiveness. As illustratedin FIG. 1, domain 9 of FcRH5 is an example of a unique sequence amongthe three isoforms. Next, there was a need for little to nocross-reactivity with FcRH family members other than FcRH5, such asFcRH1, FcRH2, FcRH3, and FcRH4. This is difficult because of thegenerally highly conserved nature of the last Ig-like domains in many ofthe FcRH family members. But because of the parallel need for FcRH5isoform c specificity, an antibody that binds the last Ig-like domainwas pursued. Finally, for antibodies to be used in therapeutic moleculesthat work to bring large structures in close proximity, such as T-cellsand tumor cells using a bispecific antibody format, it is known thattumor epitopes closer to the cell membrane are more effective (see,e.g., Bluemel et al. Cancer Immunol Immunother. (2010) 59:1197-1209).Sometimes described as the theory of kinetic segregation, the cellmembrane proximal location of domain 9 of FcRH5 is a desirable antigentarget in this context. To meet these considerations and as described indetail below, certain embodiments of the antibodies of the presentinvention were developed.

Provided herein are isolated anti-FcRH5 antibodies that binds an isoformc-specific region of the extracellular domain of FcRH5c. In someembodiments, the isoform c-specific region comprises Ig-like domain 9.In some embodiments, the Ig-like domain 9 is also called Ig-like C2-type8. In some embodiments, the isoform c-specific region comprises aminoacids 754-835 of SEQ ID NO:1. In some embodiments, the isoformc-specific region comprises amino acids 752-834 of SEQ ID NO:1. In someembodiments, the isoform c-specific region comprises amino acids 743-850of SEQ ID NO:1. In some embodiments, the isoform c-specific regioncomprises amino acids 745-851 of SEQ ID NO:1. In some embodiments, theisoform c-specific region comprises amino acids about any of 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 from the N-terminal and/orC-terminal boundary. In some embodiments, the isoform c-specific regioncomprises amino acids from about any of 750, 751, 752, 753, or 754 toabout any of 830, 831, 832, 833, 834, 835, or 836 of SEQ ID NO:1. Insome embodiments, the antibodies binds FcRH5c and/or the isoformc-specific region with an affinity of ≦5 nM, or ≦4 nM, or ≦3 nM, or ≦2nM, or ≦1 nM, and optionally ≧0.0001 nM, or ≧0.001 nM, or ≧0.01 nM.

In some embodiments of any of the antibodies, the antibody has one ormore of the following characteristics: a) cross reactive with fulllength human and cyno FcRH5 (i.e., binds full length human FcRH5 andbinds full length cyno FcRH5), b) does not significantly cross reactwith FcRH1, FcRH2, FcRH3, and/or FcRH4 (i.e., does not significantlybind FcRH1, FcRH2, FcRH3, and/or FcRH4), c) binds to endogenous FcRH5,d) does not cross react with FcRH5a (i.e., does not significantly bindFcRH5a), and e) does not cross react with another Ig-like domain ofFcRH5 (i.e., does not significantly bind another Ig-like domain ofFcRH5). Methods of determining the ability to bind are known in the artand described below.

Provided herein, and in some embodiments, are antibodies comprising a) aheavy chain comprising a HVR-H1 comprising the amino acid sequence ofSEQ ID NO:38, HVR-H2 comprising the amino acid sequence of SEQ ID NO:62,and HVR-H3 comprising the amino acid sequence of SEQ ID NO:86; and/or b)a light chain comprising a HVR-L1 comprising the amino acid sequence ofSEQ ID NO:2, HVR-L2 comprising the amino acid sequence of SEQ ID NO:14,and HVR-L3 comprising the amino acid sequence of SEQ ID NO:26. In someembodiments, the heavy chain comprising a HVR-H1 comprising the aminoacid sequence of SEQ ID NO:50, HVR-H2 comprising the amino acid sequenceof SEQ ID NO:74, and HVR-H3 comprising the amino acid sequence of SEQ IDNO:98. In some embodiments, the antibody comprises a VH sequence havingat least about any of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100% sequence identity to the amino acid sequence of SEQ ID NO:111and/or a VL sequence having at least about any of 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the aminoacid sequence of SEQ ID NO: 110. In some embodiments, the antibodycomprises a VH sequence of SEQ ID NO:111 and/or a VL sequence of SEQ IDNO:110. In some embodiments of any of the antibodies, the antibodycomprises six HVRs of 1C8.1. In some embodiments, the antibody comprisesVH domain and VL domain of 1C8.1. In some embodiments, the antibodybinds an isoform c-specific region of the extracellular domain of FcRH5c(e.g., Ig-like domain 9). In some embodiments, the antibody is crossreactive with full length human and cyno FcRH5. In some embodiments, theantibody does not significantly cross react with FcRH1, FcRH2, FcRH3,and/or FcRH4. In some embodiments, the antibody binds to endogenousFcRH5. In some embodiments, the antibody binds B-cells. In someembodiments, the antibody does not significantly bind NK cells and/ormonocytes.

Provided herein, and in some embodiments, are antibodies comprising a) aheavy chain comprising a HVR-H1 comprising the amino acid sequence ofSEQ ID NO:39, HVR-H2 comprising the amino acid sequence of SEQ ID NO:63,and HVR-H3 comprising the amino acid sequence of SEQ ID NO:87; and/or b)a light chain comprising a HVR-L1 comprising the amino acid sequence ofSEQ ID NO:3, HVR-L2 comprising the amino acid sequence of SEQ ID NO:15,and HVR-L3 comprising the amino acid sequence of SEQ ID NO:27. In someembodiments, the heavy chain comprising a HVR-H1 comprising the aminoacid sequence of SEQ ID NO:51, HVR-H2 comprising the amino acid sequenceof SEQ ID NO:75, and HVR-H3 comprising the amino acid sequence of SEQ IDNO:99. In some embodiments, the antibody comprises a VH sequence havingat least about any of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100% sequence identity to the amino acid sequence of SEQ ID NO:113and/or a VL sequence having at least about any of 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the aminoacid sequence of SEQ ID NO:112. In some embodiments, the antibodycomprises a VH sequence having at least about any of 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the aminoacid sequence of SEQ ID NO:135 and/or a VL sequence having at leastabout any of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO:134. In someembodiments, the antibody comprises a VH sequence of SEQ ID NO:113and/or a VL sequence of SEQ ID NO:112. In some embodiments, the antibodycomprises a VH sequence of SEQ ID NO:135 and/or a VL sequence of SEQ IDNO:134. In some embodiments of any of the antibodies, the antibodycomprises six HVRs of 1G7.2. In some embodiments, the antibody comprisesVH domain and VL domain of 1G7.2. In some embodiments of any of theantibodies, the antibody comprises six HVRs of 1G7.2′. In someembodiments, the antibody comprises VH domain and VL domain of 1G7.2′.In some embodiments, the antibody binds an isoform c-specific region ofthe extracellular domain of FcRH5c (e.g., Ig-like domain 9). In someembodiments, the antibody is cross reactive with full length human andcyno FcRH5. In some embodiments, the antibody does not significantlycross react with FcRH1, FcRH2, FcRH3, and/or FcRH4. In some embodiments,the antibody binds to endogenous FcRH5. In some embodiments, theantibody binds B-cells. In some embodiments, the antibody does notsignificantly bind NK cells and/or monocytes. In some embodiments, theantibody does not significantly cross react with FcRH5a.

Provided herein, and in some embodiments, are antibodies comprising a) aheavy chain comprising a HVR-H1 comprising the amino acid sequence ofSEQ ID NO:40, HVR-H2 comprising the amino acid sequence of SEQ ID NO:64,and HVR-H3 comprising the amino acid sequence of SEQ ID NO:88; and/or b)a light chain comprising a HVR-L1 comprising the amino acid sequence ofSEQ ID NO:4, HVR-L2 comprising the amino acid sequence of SEQ ID NO:16,and HVR-L3 comprising the amino acid sequence of SEQ ID NO:28. In someembodiments, the heavy chain comprising a HVR-H1 comprising the aminoacid sequence of SEQ ID NO:52, HVR-H2 comprising the amino acid sequenceof SEQ ID NO:76, and HVR-H3 comprising the amino acid sequence of SEQ IDNO:100. In some embodiments, the antibody comprises a VH sequence havingat least about any of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100% sequence identity to the amino acid sequence of SEQ ID NO:115and/or a VL sequence having at least about any of 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the aminoacid sequence of SEQ ID NO:114. In some embodiments, the antibodycomprises a VH sequence of SEQ ID NO:115 and/or a VL sequence of SEQ IDNO:114. In some embodiments of any of the antibodies, the antibodycomprises six HVRs of 2H7.3. In some embodiments, the antibody comprisesVH domain and VL domain of 2H7.3. In some embodiments, the antibodybinds an isoform c-specific region of the extracellular domain of FcRH5c(e.g., Ig-like domain 9). In some embodiments, the antibody is crossreactive with full length human and cyno FcRH5. In some embodiments, theantibody does not significantly cross react with FcRH1, FcRH3, and/orFcRH4. In some embodiments, the antibody binds to endogenous FcRH5. Insome embodiments, the antibody binds B-cells. In some embodiments, theantibody does not significantly bind NK cells and/or monocytes.

Provided herein, and in some embodiments, are antibodies comprising a) aheavy chain comprising a HVR-H1 comprising the amino acid sequence ofSEQ ID NO:41, HVR-H2 comprising the amino acid sequence of SEQ ID NO:65,and HVR-H3 comprising the amino acid sequence of SEQ ID NO:89; and/or b)a light chain comprising a HVR-L1 comprising the amino acid sequence ofSEQ ID NO:5, HVR-L2 comprising the amino acid sequence of SEQ ID NO:17,and HVR-L3 comprising the amino acid sequence of SEQ ID NO:29. In someembodiments, the heavy chain comprising a HVR-H1 comprising the aminoacid sequence of SEQ ID NO:53, HVR-H2 comprising the amino acid sequenceof SEQ ID NO:77, and HVR-H3 comprising the amino acid sequence of SEQ IDNO:101. In some embodiments, the antibody comprises a VH sequence havingat least about any of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100% sequence identity to the amino acid sequence of SEQ ID NO:117and/or a VL sequence having at least about any of 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the aminoacid sequence of SEQ ID NO:116. In some embodiments, the antibodycomprises a VH sequence of SEQ ID NO:117 and/or a VL sequence of SEQ IDNO:116. In some embodiments of any of the antibodies, the antibodycomprises six HVRs of 3A4.2. In some embodiments, the antibody comprisesVH domain and VL domain of 3A4.2. In some embodiments, the antibodybinds an isoform c-specific region of the extracellular domain of FcRH5c(e.g., Ig-like domain 9). In some embodiments, the antibody is crossreactive with full length human and cyno FcRH5. In some embodiments, theantibody does not significantly cross react with FcRH1, FcRH2, FcRH3,and/or FcRH4. In some embodiments, the antibody binds to endogenousFcRH5. In some embodiments, the antibody binds B-cells. In someembodiments, the antibody does not significantly bind NK cells and/ormonocytes.

Provided herein, and in some embodiments, are antibodies comprising a) aheavy chain comprising a HVR-H1 comprising the amino acid sequence ofSEQ ID NO:42, HVR-H2 comprising the amino acid sequence of SEQ ID NO:66,and HVR-H3 comprising the amino acid sequence of SEQ ID NO:90; and/or b)a light chain comprising a HVR-L1 comprising the amino acid sequence ofSEQ ID NO:6, HVR-L2 comprising the amino acid sequence of SEQ ID NO:18,and HVR-L3 comprising the amino acid sequence of SEQ ID NO:30. In someembodiments, the heavy chain comprising a HVR-H1 comprising the aminoacid sequence of SEQ ID NO:54, HVR-H2 comprising the amino acid sequenceof SEQ ID NO:78, and HVR-H3 comprising the amino acid sequence of SEQ IDNO:102. In some embodiments, the antibody comprises a VH sequence havingat least about any of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100% sequence identity to the amino acid sequence of SEQ ID NO:119and/or a VL sequence having at least about any of 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the aminoacid sequence of SEQ ID NO:118. In some embodiments, the antibodycomprises a VH sequence of SEQ ID NO:119 and/or a VL sequence of SEQ IDNO:118. In some embodiments of any of the antibodies, the antibodycomprises six HVRs of 3B12.1.1. In some embodiments, the antibodycomprises VH domain and VL domain of 3B12.1.1. In some embodiments, theantibody binds an isoform c-specific region of the extracellular domainof FcRH5c (e.g., Ig-like domain 9). In some embodiments, the antibody iscross reactive with full length human and cyno FcRH5. In someembodiments, the antibody does not significantly cross react with FcRH1,FcRH2, FcRH3, and/or FcRH4. In some embodiments, the antibody binds toendogenous FcRH5. In some embodiments, the antibody binds B-cells. Insome embodiments, the antibody does not significantly bind NK cellsand/or monocytes. In some embodiments, the antibody does notsignificantly cross react with FcRH5a.

Provided herein, and in some embodiments, are antibodies comprising a) aheavy chain comprising a HVR-H1 comprising the amino acid sequence ofSEQ ID NO:43, HVR-H2 comprising the amino acid sequence of SEQ ID NO:67,and HVR-H3 comprising the amino acid sequence of SEQ ID NO:91; and/or b)a light chain comprising a HVR-L1 comprising the amino acid sequence ofSEQ ID NO:7, HVR-L2 comprising the amino acid sequence of SEQ ID NO:19,and HVR-L3 comprising the amino acid sequence of SEQ ID NO:31. In someembodiments, the heavy chain comprising a HVR-H1 comprising the aminoacid sequence of SEQ ID NO:55, HVR-H2 comprising the amino acid sequenceof SEQ ID NO:79, and HVR-H3 comprising the amino acid sequence of SEQ IDNO:103. In some embodiments, the antibody comprises a VH sequence havingat least about any of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100% sequence identity to the amino acid sequence of SEQ ID NO:121and/or a VL sequence having at least about any of 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the aminoacid sequence of SEQ ID NO:120. In some embodiments, the antibodycomprises a VH sequence of SEQ ID NO:121 and/or a VL sequence of SEQ IDNO:120. In some embodiments of any of the antibodies, the antibodycomprises six HVRs of 3C10. In some embodiments, the antibody comprisesVH domain and VL domain of 3C10. In some embodiments, the antibody bindsan isoform c-specific region of the extracellular domain of FcRH5c(e.g., Ig-like domain 9). In some embodiments, the antibody is crossreactive with full length human and cyno FcRH5. In some embodiments, theantibody does not significantly cross react with FcRH1, FcRH2, FcRH3,and/or FcRH4. In some embodiments, the antibody binds to endogenousFcRH5. In some embodiments, the antibody binds B-cells. In someembodiments, the antibody does not significantly bind NK cells and/ormonocytes.

Provided herein, and in some embodiments, are antibodies comprising a) aheavy chain comprising a HVR-H1 comprising the amino acid sequence ofSEQ ID NO:44, HVR-H2 comprising the amino acid sequence of SEQ ID NO:68,and HVR-H3 comprising the amino acid sequence of SEQ ID NO:92; and/or b)a light chain comprising a HVR-L1 comprising the amino acid sequence ofSEQ ID NO:8, HVR-L2 comprising the amino acid sequence of SEQ ID NO:20,and HVR-L3 comprising the amino acid sequence of SEQ ID NO:32. In someembodiments, the heavy chain comprising a HVR-H1 comprising the aminoacid sequence of SEQ ID NO:56, HVR-H2 comprising the amino acid sequenceof SEQ ID NO:80, and HVR-H3 comprising the amino acid sequence of SEQ IDNO:104. In some embodiments, the antibody comprises a VH sequence havingat least about any of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100% sequence identity to the amino acid sequence of SEQ ID NO:123and/or a VL sequence having at least about any of 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the aminoacid sequence of SEQ ID NO:122. In some embodiments, the antibodycomprises a VH sequence of SEQ ID NO:123 and/or a VL sequence of SEQ IDNO:122. In some embodiments of any of the antibodies, the antibodycomprises six HVRs of 3F10. In some embodiments, the antibody comprisesVH domain and VL domain of 3F10. In some embodiments, the antibody bindsan isoform c-specific region of the extracellular domain of FcRH5c(e.g., Ig-like domain 9). In some embodiments, the antibody is crossreactive with full length human and cyno FcRH5. In some embodiments, theantibody does not significantly cross react with FcRH1, FcRH2, FcRH3,and/or FcRH4. In some embodiments, the antibody binds to endogenousFcRH5. In some embodiments, the antibody binds B-cells. In someembodiments, the antibody does not significantly bind NK cells and/ormonocytes. In some embodiments, the antibody does not significantlycross react with FcRH5a.

Provided herein, and in some embodiments, are antibodies comprising a) aheavy chain comprising a HVR-H1 comprising the amino acid sequence ofSEQ ID NO:45, HVR-H2 comprising the amino acid sequence of SEQ ID NO:69,and HVR-H3 comprising the amino acid sequence of SEQ ID NO:93; and/or b)a light chain comprising a HVR-L1 comprising the amino acid sequence ofSEQ ID NO:9, HVR-L2 comprising the amino acid sequence of SEQ ID NO:21,and HVR-L3 comprising the amino acid sequence of SEQ ID NO:33. In someembodiments, the heavy chain comprising a HVR-H1 comprising the aminoacid sequence of SEQ ID NO:57, HVR-H2 comprising the amino acid sequenceof SEQ ID NO:81, and HVR-H3 comprising the amino acid sequence of SEQ IDNO:105. In some embodiments, the antibody comprises a VH sequence havingat least about any of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100% sequence identity to the amino acid sequence of SEQ ID NO:125and/or a VL sequence having at least about any of 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the aminoacid sequence of SEQ ID NO:124. In some embodiments, the antibodycomprises a VH sequence of SEQ ID NO:125 and/or a VL sequence of SEQ IDNO:124. In some embodiments of any of the antibodies, the antibodycomprises six HVRs of 3G3. In some embodiments, the antibody comprisesVH domain and VL domain of 3G3. In some embodiments, the antibody bindsan isoform c-specific region of the extracellular domain of FcRH5c(e.g., Ig-like domain 9). In some embodiments, the antibody is crossreactive with full length human and cyno FcRH5. In some embodiments, theantibody does not significantly cross react with FcRH1, FcRH2, FcRH3,and/or FcRH4. In some embodiments, the antibody binds to endogenousFcRH5. In some embodiments, the antibody binds B-cells. In someembodiments, the antibody does not significantly bind NK cells and/ormonocytes. In some embodiments, the antibody does not significantlycross react with FcRH5a.

Provided herein, and in some embodiments, are antibodies comprising a) aheavy chain comprising a HVR-H1 comprising the amino acid sequence ofSEQ ID NO:46, HVR-H2 comprising the amino acid sequence of SEQ ID NO:70,and HVR-H3 comprising the amino acid sequence of SEQ ID NO:94; and/or b)a light chain comprising a HVR-L1 comprising the amino acid sequence ofSEQ ID NO:10, HVR-L2 comprising the amino acid sequence of SEQ ID NO:22,and HVR-L3 comprising the amino acid sequence of SEQ ID NO:34. In someembodiments, the heavy chain comprising a HVR-H1 comprising the aminoacid sequence of SEQ ID NO:58, HVR-H2 comprising the amino acid sequenceof SEQ ID NO:82, and HVR-H3 comprising the amino acid sequence of SEQ IDNO:106. In some embodiments, the antibody comprises a VH sequence havingat least about any of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100% sequence identity to the amino acid sequence of SEQ ID NO:127and/or a VL sequence having at least about any of 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the aminoacid sequence of SEQ ID NO: 126. In some embodiments, the antibodycomprises a VH sequence of SEQ ID NO:127 and/or a VL sequence of SEQ IDNO:126. In some embodiments of any of the antibodies, the antibodycomprises six HVRs of 3G7.1.5. In some embodiments, the antibodycomprises VH domain and VL domain of 3G7.1.5. In some embodiments, theantibody binds an isoform c-specific region of the extracellular domainof FcRH5c (e.g., Ig-like domain 9). In some embodiments, the antibody iscross reactive with full length human and cyno FcRH5. In someembodiments, the antibody does not significantly cross react with FcRH1,FcRH2, FcRH3, and/or FcRH4. In some embodiments, the antibody binds toendogenous FcRH5. In some embodiments, the antibody binds B-cells. Insome embodiments, the antibody does not significantly bind NK cellsand/or monocytes.

Provided herein, and in some embodiments, are antibodies comprising a) aheavy chain comprising a HVR-H1 comprising the amino acid sequence ofSEQ ID NO:47, HVR-H2 comprising the amino acid sequence of SEQ ID NO:71,and HVR-H3 comprising the amino acid sequence of SEQ ID NO:95; and/or b)a light chain comprising a HVR-L1 comprising the amino acid sequence ofSEQ ID NO:11, HVR-L2 comprising the amino acid sequence of SEQ ID NO:23,and HVR-L3 comprising the amino acid sequence of SEQ ID NO:35. In someembodiments, the heavy chain comprising a HVR-H1 comprising the aminoacid sequence of SEQ ID NO:59, HVR-H2 comprising the amino acid sequenceof SEQ ID NO:83, and HVR-H3 comprising the amino acid sequence of SEQ IDNO:107. In some embodiments, the antibody comprises a VH sequence havingat least about any of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100% sequence identity to the amino acid sequence of SEQ ID NO:129and/or a VL sequence having at least about any of 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the aminoacid sequence of SEQ ID NO:128. In some embodiments, the antibodycomprises a VH sequence of SEQ ID NO:129 and/or a VL sequence of SEQ IDNO:128. In some embodiments of any of the antibodies, the antibodycomprises six HVRs of 5A10.1.3. In some embodiments, the antibodycomprises VH domain and VL domain of 5A10.1.3. In some embodiments, theantibody binds an isoform c-specific region of the extracellular domainof FcRH5c (e.g., Ig-like domain 9). In some embodiments, the antibody iscross reactive with full length human and cyno FcRH5. In someembodiments, the antibody does not significantly cross react with FcRH1,FcRH2, FcRH3, and/or FcRH4. In some embodiments, the antibody binds toendogenous FcRH5. In some embodiments, the antibody binds B-cells. Insome embodiments, the antibody does not significantly bind NK cellsand/or monocytes.

Provided herein, and in some embodiments, are antibodies comprising a) aheavy chain comprising a HVR-H1 comprising the amino acid sequence ofSEQ ID NO:48, HVR-H2 comprising the amino acid sequence of SEQ ID NO:72,and HVR-H3 comprising the amino acid sequence of SEQ ID NO:96; and/or b)a light chain comprising a HVR-L1 comprising the amino acid sequence ofSEQ ID NO:12, HVR-L2 comprising the amino acid sequence of SEQ ID NO:24,and HVR-L3 comprising the amino acid sequence of SEQ ID NO:36. In someembodiments, the heavy chain comprising a HVR-H1 comprising the aminoacid sequence of SEQ ID NO:60, HVR-H2 comprising the amino acid sequenceof SEQ ID NO:84, and HVR-H3 comprising the amino acid sequence of SEQ IDNO:108. In some embodiments, the antibody comprises a VH sequence havingat least about any of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100% sequence identity to the amino acid sequence of SEQ ID NO:131and/or a VL sequence having at least about any of 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the aminoacid sequence of SEQ ID NO:130. In some embodiments, the antibodycomprises a VH sequence of SEQ ID NO:131 and/or a VL sequence of SEQ IDNO:130. In some embodiments of any of the antibodies, the antibodycomprises six HVRs of 5F1.1.5. In some embodiments, the antibodycomprises VH domain and VL domain of 5F1.1.5. In some embodiments, theantibody binds an isoform c-specific region of the extracellular domainof FcRH5c (e.g., Ig-like domain 9). In some embodiments, the antibody iscross reactive with full length human and cyno FcRH5. In someembodiments, the antibody does not significantly cross react with FcRH1,FcRH2, FcRH3, and/or FcRH4. In some embodiments, the antibody binds toendogenous FcRH5. In some embodiments, the antibody binds B-cells. Insome embodiments, the antibody does not significantly bind NK cellsand/or monocytes. In some embodiments, the antibody does notsignificantly cross react with FcRH5a.

Provided herein, and in some embodiments, are antibodies comprising a) aheavy chain comprising a HVR-H1 comprising the amino acid sequence ofSEQ ID NO:49, HVR-H2 comprising the amino acid sequence of SEQ ID NO:73,and HVR-H3 comprising the amino acid sequence of SEQ ID NO:97; and/or b)a light chain comprising a HVR-L1 comprising the amino acid sequence ofSEQ ID NO:13, HVR-L2 comprising the amino acid sequence of SEQ ID NO:25,and HVR-L3 comprising the amino acid sequence of SEQ ID NO:37. In someembodiments, the heavy chain comprising a HVR-H1 comprising the aminoacid sequence of SEQ ID NO:61, HVR-H2 comprising the amino acid sequenceof SEQ ID NO:85, and HVR-H3 comprising the amino acid sequence of SEQ IDNO:109. In some embodiments, the antibody comprises a VH sequence havingat least about any of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100% sequence identity to the amino acid sequence of SEQ ID NO:133and/or a VL sequence having at least about any of 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the aminoacid sequence of SEQ ID NO:132. In some embodiments, the antibodycomprises a VH sequence of SEQ ID NO:133 and/or a VL sequence of SEQ IDNO:132. In some embodiments of any of the antibodies, the antibodycomprises six HVRs of 6D2. In some embodiments, the antibody comprisesVH domain and VL domain of 6D2. In some embodiments, the antibody bindsan isoform c-specific region of the extracellular domain of FcRH5c(e.g., Ig-like domain 9). In some embodiments, the antibody is crossreactive with full length human and cyno FcRH5. In some embodiments, theantibody does not significantly cross react with FcRH1, FcRH2, FcRH3,and/or FcRH4. In some embodiments, the antibody binds to endogenousFcRH5. In some embodiments, the antibody binds B-cells. In someembodiments, the antibody does not significantly bind NK cells and/ormonocytes.

In a further aspect provided herein, an anti-FcRH5 antibody according toany of the above embodiments is a monoclonal antibody, including achimeric, humanized or human antibody. In one embodiment, an anti-FcRH5antibody is an antibody fragment, e.g., a Fv, Fab, Fab′, scFv, diabody,or F(ab′)₂ fragment. In another embodiment, the antibody is asubstantially full length antibody, e.g., an IgG1 antibody or otherantibody class or isotype as defined herein.

In a further aspect, the invention provides an antibody that binds tothe same epitope as an anti-FcRH5 antibody provided herein. In certainembodiments, an antibody is provided that binds an isoform c-specificregion of the extracellular domain of FcRH5c from, within, oroverlapping amino acids 754-835 of SEQ ID NO:1.

In some embodiments of any of the anti-FcRH5 antibodies, the FcRH5antibody, particularly an FcRH5 bispecific (e.g., anti-CD3/anti-FcRH5bispecific), may have features, singly or in combination, based upon HEKcell line assays (HEK cells reconstituted with necessary signalingcomponents for the TCR triggering as described in James and Valle,Nature 487:64-69 (2012), which is incorporated by reference in itsentirety. In some embodiments, the features, singly or in combination,may include tumor cell interphase/immunological synapse, Lck-mediatedTCR phosphorylation, ZAP70 activity including phosphorylation state andlocalization, CD58 activity including localization and binding, β₂Aractivity including localization and binding, CAAX activity includinglocalization and binding CD45 activity including localization, pMHCactivity including localization, and/or TCR activity and triggeringfeatures.

In a further aspect, an anti-FcRH5 antibody according to any of theabove embodiments may incorporate any of the features, singly or incombination, as described in (a)-(e) and/or Sections 1-7 below.

(a) Binds an Isoform c-Specific Region of the Extracellular Domain ofFcRH5c

Methods of determining whether an anti-FcRH5 antibody binds to anisoform c-specific region of the extracellular domain of FcRH5c areknown in the art. In some embodiments, binding of an anti-FcRH5 antibodyto an isoform c-specific region of the extracellular domain of FcRH5cmay be determined by expressing FcRH5 polypeptides with N- andC-terminal deletions in 293 cells and/or SVT2 cells and testing by FACSas described in the Examples binding of the antibody to the truncatedpolypeptides. In some embodiments, a substantial reduction (≧70%reduction) or elimination of binding of the antibody to a truncatedpolypeptide relative to binding to full-length FcRH5 expressed in 293cells indicates that the antibody does not bind to that truncatedpolypeptide.

In some embodiments, the isoform c-specific region comprises Ig-likedomain 9. In some embodiments, the Ig-like domain 9 is also calledIg-like C2-type 8. In some embodiments, the isoform c-specific regioncomprises amino acids 754-835 of SEQ ID NO:1. In some embodiments, theisoform c-specific region comprises amino acids 752-834 of SEQ ID NO:1.In some embodiments, the isoform c-specific region comprises amino acids743-850 of SEQ ID NO:1. In some embodiments, the isoform c-specificregion comprises amino acids 745-851 of SEQ ID NO:1. In someembodiments, the isoform c-specific region comprises amino acids aboutany of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 from theN-terminal and/or C-terminal boundary. In some embodiments, the isoformc-specific region comprises amino acids from about any of 750, 751, 752,753, or 754 to about any of 830, 831, 832, 833, 834, 835, or 836 of SEQID NO:1. In some embodiments, FcRH5 is human FcRH5. In some embodiments,FcRH5 is human FcRH5 or cynomolgus monkey FcRH5.

(b) Cross Reacts with (Binds) Human and Cyno FcRH5 with an Affinity of≦5 nM, or ≦4 nM, or ≦3 nM, or ≦2 nM, or ≦1 nM, and Optionally ≧0.0001nM, or ≧0.001 nM, or ≧0.01 nM

Methods of determining binding affinity are known in the art. In someembodiments, the binding affinity may be determined according to aBIAcore® assay, ELISA, Facs, and IHC, for example, as described in theExamples.

In some embodiments, the anti-FcRH5 antibody binds human and/or cynoFcRH5 with an affinity of about any of ≦5 nM, or ≦4 nM, or ≦3 nM, or ≦2nM, or ≦1 nM. In some embodiments, the anti-FcRH5 antibody binds humanand/or cyno FcRH5 with an affinity of about ≦5. In some embodiments, theanti-FcRH5 antibody binds human and/or cyno FcRH5 with an affinity ofabout ≦4 nM. In some embodiments, the anti-FcRH5 antibody binds humanand/or cyno FcRH5 with an affinity of about ≦3 nM. In some embodiments,the anti-FcRH5 antibody binds human and/or cyno FcRH5 with an affinityof about ≦2 nM. In some embodiments, FcRH5 is human FcRH5. In someembodiments, FcRH5 is cynomolgus monkey FcRH5.

(c) does not Cross React with (does not Bind) FcRH1, FcRH2, FcRH3,and/or FcRH4

Methods of determining binding are known in the art. In someembodiments, the binding affinity may be determined according to aBIAcore® assay, Facs, ELISA, and IHC, for example, as described in theExamples.

In some embodiments, the anti-FcRH5 antibody binds FcRH5 with anaffinity of more than about any of 2, 5, 10, 20, 50, 100, 500, or1000-fold greater than FcRH1, FcRH2, FcRH3, and/or FcRH4. In someembodiments, FcRH is human FcRH.

(d) does not Cross React with (does not Bind) FcRH5a

Methods of determining binding are known in the art. In someembodiments, the binding affinity may be determined according to aBIAcore® assay, Facs, ELISA, and IHC, for example, as described in theExamples.

In some embodiments, the anti-FcRH5 antibody binds FcRH5c with anaffinity of more than about any of 2, 5, 10, 20, 50, 100, 500, or1000-fold greater than FcRH5a. In some embodiments, FcRH is human FcRH.

(e) does not Cross React with Another Ig-Like Domain (does not Bind) ofFcRH5

Methods of determining binding are known in the art. In someembodiments, the binding affinity may be determined according to aBIAcore® assay, Facs, ELISA, and IHC, for example, as described in theExamples.

In some embodiments, the anti-FcRH5 antibody binds Ig-like domain 9 ofFcRH5 with an affinity of more than about any of 2, 5, 10, 20, 50, 100,500, or 1000-fold greater than Ig-like domain 1, 2, 3, 4, 5, 6, 7,and/or 8 of FcRH5. In some embodiments, FcRH is human FcRH. In someembodiments, the Ig-like domain is Ig-like domain 1 (aa 23-100 of SEQ IDNO:1), Ig-like domain 2 (aa 105-185 of SEQ ID NO:1), Ig-like domain 3(aa 188-271 of SEQ ID NO:1), Ig-like domain 4 (287-373 of SEQ ID NO:1),Ig-like domain 5 (aa 380-466 of SEQ ID NO:1), Ig-like domain 6 (aa490-555 of SEQ ID NO:1), Ig-like domain 7 (aa 568-652 of SEQ ID NO:1),Ig-like domain 8 (aa 658-731 of SEQ ID NO:1).

Binding Assays and Other Assays

In one aspect, an anti-FcRH5 antibody is tested for its antigen bindingactivity. For example, in certain embodiments, an anti-FcRH5 antibody istested for its ability to bind to FcRH5 expressed on the surface of acell. A FACS assay may be used for such testing.

In an exemplary competition assay, immobilized FcRH5 is incubated in asolution comprising a first labeled antibody that binds to FcRH5 and asecond unlabeled antibody that is being tested for its ability tocompete with the first antibody for binding to FcRH5. The secondantibody may be present in a hybridoma supernatant. As a control,immobilized FcRH5 is incubated in a solution comprising the firstlabeled antibody but not the second unlabeled antibody. After incubationunder conditions permissive for binding of the first antibody to FcRH5,excess unbound antibody is removed, and the amount of label associatedwith immobilized FcRH5 is measured. If the amount of label associatedwith immobilized FcRH5 is substantially reduced in the test samplerelative to the control sample, then that indicates that the secondantibody is competing with the first antibody for binding to FcRH5. Incertain embodiments, immobilized FcRH5 is present on the surface of acell or in a membrane preparation obtained from a cell expressing FcRH5on its surface.

In one aspect, purified anti-FcRH5 antibodies can be furthercharacterized by a series of assays including, but not limited to,N-terminal sequencing, amino acid analysis, non-denaturing sizeexclusion high pressure liquid chromatography (HPLC), mass spectrometry,ion exchange chromatography and papain digestion. In one embodiment,contemplated are an altered antibody that possesses some but not alleffector functions, which make it a desirable candidate for manyapplications in which the half life of the antibody in vivo is importantyet certain effector functions (such as complement and ADCC) areunnecessary or deleterious. In certain embodiments, the Fc activities ofthe antibody are measured to ensure that only the desired properties aremaintained. In vitro and/or in vivo cytotoxicity assays can be conductedto confirm the reduction/depletion of CDC and/or ADCC activities. Forexample, Fc receptor (FcR) binding assays can be conducted to ensurethat the antibody lacks FcγR binding (hence likely lacking ADCCactivity), but retains FcRn binding ability. The primary cells formediating ADCC, NK cells, express Fc(RIII only, whereas monocytesexpress Fc(RI, Fc(RII and Fc(RIII. FcR expression on hematopoietic cellsis summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev.Immunol 9:457-92 (1991). An example of an in vitro assay to assess ADCCactivity of a molecule of interest is described in U.S. Pat. No.5,500,362 or 5,821,337. Useful effector cells for such assays includeperipheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells.Alternatively, or additionally, ADCC activity of the molecule ofinterest may be assessed in vivo, e.g., in a animal model such as thatdisclosed in Clynes et al. PNAS (USA) 95:652-656 (1998). C1q bindingassays may also be carried out to confirm that the antibody is unable tobind C1q and hence lacks CDC activity. To assess complement activation,a CDC assay, e.g. as described in Gazzano-Santoro et al., J. ImmunolMethods 202:163 (1996), may be performed. FcRn binding and in vivoclearance/half life determinations can also be performed using methodsknown in the art.

1. Antibody Affinity

In certain embodiments, an antibody provided herein has a dissociationconstant (Kd) of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or≦0.001 nM, and optionally is ≧10⁻¹³ M. (e.g. 10⁻⁸M or less, e.g. from10⁻⁸M to 10⁻¹³M, e.g., from 10⁻⁹M to 10⁻¹³ M).

In some embodiments, Kd may be measured by a radiolabeled antigenbinding assay (RIA) performed with the Fab version of an antibody ofinterest and its antigen as described by the following assay.

Solution binding affinity of Fabs for antigen may be measured byequilibrating Fab with a minimal concentration of (¹²⁵I)-labeled antigenin the presence of a titration series of unlabeled antigen, thencapturing bound antigen with an anti-Fab antibody-coated plate (see,e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999)). To establishconditions for the assay, MICROTITER® multi-well plates (ThermoScientific) may be coated overnight with 5 μg/ml of a capturing anti-Fabantibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), andsubsequently blocked with 2% (w/v) bovine serum albumin in PBS for twoto five hours at room temperature (approximately 23° C.). In anon-adsorbent plate (Nunc #269620), 100 pM or 26 pM [¹²⁵I]-antigen aremixed with serial dilutions of a Fab of interest (e.g., consistent withassessment of the anti-VEGF antibody, Fab-12, in Presta et al., CancerRes. 57:4593-4599 (1997)). The Fab of interest is then incubatedovernight; however, the incubation may continue for a longer period(e.g., about 65 hours) to ensure that equilibrium is reached.Thereafter, the mixtures may be transferred to the capture plate forincubation at room temperature (e.g., for one hour). The solution may bethen removed and the plate washed eight times with 0.1% polysorbate 20(TWEEN-20®) in PBS. When the plates have dried, 150 μL/well ofscintillant (MICROSCINT-20™; Packard) may be added, and the plates maybe counted on a TOPCOUNT™ gamma counter (Packard) for ten minutes.Concentrations of each Fab that give less than or equal to 20% ofmaximal binding may be chosen for use in competitive binding assays.

According to another embodiment, Kd is measured using surface plasmonresonance assays using a BIACORE®-2000 or a BIACORE®-3000 (BIAcore,Inc., Piscataway, N.J.) at 25° C. with immobilized antigen CM5 chips at˜10 response units (RU). Briefly, carboxymethylated dextran biosensorchips (CM5, BIACORE, Inc.) may be activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions.Antigen may be diluted with 10 mM sodium acetate, pH 4.8, to 5 μg/ml(˜0.2 μM) before injection at a flow rate of 5 μL/minute to achieveapproximately 10 response units (RU) of coupled protein. Following theinjection of antigen, 1 M ethanolamine may be injected to blockunreacted groups. For kinetics measurements, two-fold serial dilutionsof Fab (0.78 nM to 500 nM) may be injected in PBS with 0.05% polysorbate20 (TWEEN-20™) surfactant (PBST) at 25° C. at a flow rate ofapproximately 25 μL/min. Association rates (k_(on)) and dissociationrates (k_(off)) may be calculated using a simple one-to-one Langmuirbinding model (BIACORE® Evaluation Software version 3.2) bysimultaneously fitting the association and dissociation sensorgrams. Theequilibrium dissociation constant (Kd) may be calculated as the ratiok_(off)/k_(on). See, e.g., Chen et al., J. Mol. Biol. 293:865-881(1999). If the on-rate exceeds 10⁶ M⁻¹s⁻¹ by the surface plasmonresonance assay above, then the on-rate may be determined by using afluorescent quenching technique that measures the increase or decreasein fluorescence emission intensity (excitation=295 nm; emission=340 nm,16 nm band-pass) at 25° C. of a 20 nM anti-antigen antibody (Fab form)in PBS, pH 7.2, in the presence of increasing concentrations of antigenas measured in a spectrometer, such as a stop-flow equippedspectrophometer (Aviv Instruments) or a 8000-series SLM-AMINCO™spectrophotometer (ThermoSpectronic) with a stirred cuvette.

2. Antibody Fragments

In certain embodiments, an antibody provided herein is an antibodyfragment. Antibody fragments include, but are not limited to, Fab, Fab′,Fab′-SH, F(ab′)₂, Fv, and scFv fragments, and other fragments describedbelow. For a review of certain antibody fragments, see Hudson et al.Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, e.g.,Pluckthün, in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315(1994); see also WO 93/16185; and U.S. Pat. Nos. 5,571,894 and5,587,458. For discussion of Fab and F(ab′)₂ fragments comprisingsalvage receptor binding epitope residues and having increased in vivohalf-life, see U.S. Pat. No. 5,869,046.

Diabodies are antibody fragments with two antigen-binding sites that maybe bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161;Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc.Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodiesare also described in Hudson et al., Nat. Med. 9:129-134 (2003).

Single-domain antibodies are antibody fragments comprising all or aportion of the heavy chain variable domain or all or a portion of thelight chain variable domain of an antibody. In certain embodiments, asingle-domain antibody is a human single-domain antibody (Domantis,Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516 B1).

Antibody fragments can be made by various techniques, including but notlimited to proteolytic digestion of an intact antibody as well asproduction by recombinant host cells (e.g. E. coli or phage), asdescribed herein.

3. Chimeric and Humanized Antibodies

In certain embodiments, an antibody provided herein is a chimericantibody. Certain chimeric antibodies are described, e.g., in U.S. Pat.No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA,81:6851-6855 (1984)). In one example, a chimeric antibody comprises anon-human variable region (e.g., a variable region derived from a mouse,rat, hamster, rabbit, or non-human primate, such as a monkey) and ahuman constant region. In a further example, a chimeric antibody is a“class switched” antibody in which the class or subclass has beenchanged from that of the parent antibody Chimeric antibodies includeantigen-binding fragments thereof.

In certain embodiments, a chimeric antibody is a humanized antibody.Typically, a non-human antibody is humanized to reduce immunogenicity tohumans, while retaining the specificity and affinity of the parentalnon-human antibody. Generally, a humanized antibody comprises one ormore variable domains in which HVRs, e.g., CDRs, (or portions thereof)are derived from a non-human antibody, and FRs (or portions thereof) arederived from human antibody sequences. A humanized antibody optionallywill also comprise at least a portion of a human constant region. Insome embodiments, some FR residues in a humanized antibody aresubstituted with corresponding residues from a non-human antibody (e.g.,the antibody from which the HVR residues are derived), e.g., to restoreor improve antibody specificity or affinity.

Humanized antibodies and methods of making them are reviewed, e.g., inAlmagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and arefurther described, e.g., in Riechmann et al., Nature 332:323-329 (1988);Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); US Pat.Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al.,Methods 36:25-34 (2005) (describing SDR (a-CDR) grafting); Padlan, Mol.Immunol. 28:489-498 (1991) (describing “resurfacing”); Dall'Acqua etal., Methods 36:43-60 (2005) (describing “FR shuffling”); and Osbourn etal., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer,83:252-260 (2000) (describing the “guided selection” approach to FRshuffling).

Human framework regions that may be used for humanization include butare not limited to: framework regions selected using the “best-fit”method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); frameworkregions derived from the consensus sequence of human antibodies of aparticular subgroup of light or heavy chain variable regions (see, e.g.,Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta etal. J. Immunol., 151:2623 (1993)); human mature (somatically mutated)framework regions or human germline framework regions (see, e.g.,Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and frameworkregions derived from screening FR libraries (see, e.g., Baca et al., J.Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem.271:22611-22618 (1996)).

4. Human Antibodies

In certain embodiments, an antibody provided herein is a human antibody.Human antibodies can be produced using various techniques known in theart. Human antibodies are described generally in van Dijk and van deWinkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin.Immunol. 20:450-459 (2008).

Human antibodies may be prepared by administering an immunogen to atransgenic animal that has been modified to produce intact humanantibodies or intact antibodies with human variable regions in responseto antigenic challenge. Such animals typically contain all or a portionof the human immunoglobulin loci, which replace the endogenousimmunoglobulin loci, or which are present extrachromosomally orintegrated randomly into the animal's chromosomes. In such transgenicmice, the endogenous immunoglobulin loci have generally beeninactivated. For review of methods for obtaining human antibodies fromtransgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). Seealso, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 describing XENOMOUSE™technology; U.S. Pat. No. 5,770,429 describing HuMAB® technology; U.S.Pat. No. 7,041,870 describing K-M MOUSE® technology, and U.S. PatentApplication Publication No. US 2007/0061900, describing VELOCIMOUSE®technology). Human variable regions from intact antibodies generated bysuch animals may be further modified, e.g., by combining with adifferent human constant region.

Human antibodies can also be made by hybridoma-based methods. Humanmyeloma and mouse-human heteromyeloma cell lines for the production ofhuman monoclonal antibodies have been described. (See, e.g., Kozbor J.Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc.,New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991).) Humanantibodies generated via human B-cell hybridoma technology are alsodescribed in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562(2006). Additional methods include those described, for example, in U.S.Pat. No. 7,189,826 (describing production of monoclonal human IgMantibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue,26(4):265-268 (2006) (describing human-human hybridomas). Humanhybridoma technology (Trioma technology) is also described in Vollmersand Brandlein, Histology and Histopathology, 20(3):927-937 (2005) andVollmers and Brandlein, Methods and Findings in Experimental andClinical Pharmacology, 27(3):185-91 (2005).

Human antibodies may also be generated by isolating Fv clone variabledomain sequences selected from human-derived phage display libraries.Such variable domain sequences may then be combined with a desired humanconstant domain. Techniques for selecting human antibodies from antibodylibraries are described below.

5. Library-Derived Antibodies

Antibodies provided herein may be isolated by screening combinatoriallibraries for antibodies with the desired activity or activities. Forexample, a variety of methods are known in the art for generating phagedisplay libraries and screening such libraries for antibodies possessingthe desired binding characteristics. Such methods are reviewed, e.g., inHoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien etal., ed., Human Press, Totowa, N.J., 2001) and further described, e.g.,in the McCafferty et al., Nature 348:552-554; Clackson et al., Nature352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992);Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo,ed., Human Press, Totowa, N.J., 2003); Sidhu et al., J. Mol. Biol.338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093(2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472(2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132 (2004).

In certain phage display methods, repertoires of VH and VL genes areseparately cloned by polymerase chain reaction (PCR) and recombinedrandomly in phage libraries, which can then be screened forantigen-binding phage as described in Winter et al., Ann. Rev. Immunol.,12: 433-455 (1994). Phage typically display antibody fragments, eitheras single-chain Fv (scFv) fragments or as Fab fragments. Libraries fromimmunized sources provide high-affinity antibodies to the immunogenwithout the requirement of constructing hybridomas. Alternatively, thenaive repertoire can be cloned (e.g., from human) to provide a singlesource of antibodies to a wide range of non-self and also self antigenswithout any immunization as described by Griffiths et al., EMBO J, 12:725-734 (1993). Finally, naive libraries can also be made syntheticallyby cloning unrearranged V-gene segments from stem cells, and using PCRprimers containing random sequence to encode the highly variable CDR3regions and to accomplish rearrangement in vitro, as described byHoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). Patentpublications describing human antibody phage libraries include, forexample: U.S. Pat. No. 5,750,373, and US Patent Publication Nos.2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598,2007/0237764, 2007/0292936, and 2009/0002360.

Antibodies or antibody fragments isolated from human antibody librariesare considered human antibodies or human antibody fragments herein.

6. Multispecific Antibodies

In certain embodiments, an antibody provided herein is a multispecificantibody, e.g. a bispecific antibody. Multispecific antibodies aremonoclonal antibodies that have binding specificities for at least twodifferent sites. In certain embodiments, one of the bindingspecificities is for FcRH5 and the other is for any other antigen. Incertain embodiments, one of the binding specificities is for FcRH5 andthe other is for CD3. See, e.g., U.S. Pat. No. 5,821,337. In certainembodiments, bispecific antibodies may bind to two different epitopes ofFcRH5. Bispecific antibodies may also be used to localize cytotoxicagents to cells which express FcRH5. Bispecific antibodies can beprepared as full length antibodies or antibody fragments.

In some embodiments, the FcRH5 antibodies are FcRH5 bispecificantibodies. Bispecific antibodies are antibodies that have bindingspecificities for at least two different epitopes. Exemplary bispecificantibodies may bind to two different epitopes of an FcRH5 protein asdescribed herein. Other such antibodies may combine an FcRH5 bindingsite with a binding site for another protein. Alternatively, ananti-FcRH5 arm may be combined with an arm which binds to a triggeringmolecule on a leukocyte such as a T-cell receptor molecule (e.g. CD3),or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32) andFcγRIII (CD16), so as to focus and localize cellular defense mechanismsto the FcRH5-expressing cell. Bispecific antibodies may also be used tolocalize cytotoxic agents to cells which express FcRH5. These antibodiespossess an FcRH5-binding arm and an arm which binds the cytotoxic agent(e.g., saporin, anti-interferon α vinca alkaloid, ricin A chain,methotrexate or radioactive isotope hapten). Bispecific antibodies canbe prepared as full length antibodies or antibody fragments (e.g.,F(ab)₂ bispecific antibodies). In some embodiments, the anti-FcRH5antibody binds an isoform c-specific region of the extracellular domainof FcRH5c. In some embodiments, the anti-FcRH5 antibodies binds Ig-likedomain 9 of FcRH5c.

In some embodiments, the FcRH5 bispecific antibody comprises a firstarm, wherein the first arm binds FcRH5 and a second arm, wherein thesecond arm binds a Fc. The second arm of the FcRH5 bispecific antibodymay be any anti-Fc antibody known in the art. For example, WO 96/16673describes a bispecific anti-ErbB2/anti-FcγRIII antibody and U.S. Pat.No. 5,837,234 discloses a bispecific anti-ErbB2/anti-FcγRI antibody. Abispecific anti-ErbB2/Fcα antibody is shown in WO98/02463. In someembodiments, the anti-FcRH5 antibody binds an isoform c-specific regionof the extracellular domain of FcRH5c. In some embodiments, theanti-FcRH5 antibodies binds Ig-like domain 9 of FcRH5c.

In some embodiments, the FcRH5 bispecific antibody comprises a firstarm, wherein the first arm binds FcRH5 and a second arm, wherein thesecond arm binds CD3. The second arm of the FcRH5 bispecific antibodymay be any anti-CD3 antibody known in the art. U.S. Pat. Nos. 5,821,337and 6,407,213 teach bispecific anti-ErbB2/anti-CD3 antibodies.Additional bispecific antibodies that bind an epitope on the CD3 antigenand a second epitope have been described. See, for example, U.S. Pat.No. 5,078,998 (anti-CD3/tumor cell antigen); U.S. Pat. No. 5,601,819(anti-CD3/IL-2R; anti-CD3/CD28; anti-CD3/CD45); U.S. Pat. No. 6,129,914(anti-CD3/malignant B-cell antigen); U.S. Pat. No. 7,112,324(anti-CD3/CD19); U.S. Pat. No. 6,723,538 (anti-CD3/CCR5); U.S. Pat. No.7,235,641 (anti-CD3/EpCAM); U.S. Pat. No. 7,262,276 (anti-CD3/ovariantumor antigen); and U.S. Pat. No. 5,731,168 (anti-CD3/CD4IgG), which areincorporated by reference in their entirety. In some embodiments, theanti-CD3 antibody of the second arm is an antibody described in any oneof WO 2005/118635, WO2007/042261, WO2008/119567, U.S. Pat. No.5,929,212, U.S. Pat. No. 6,750,325, U.S. Pat. No. 6,491,916, U.S. Pat.No. 7,994,289, U.S. Pat. No. 7,993,641, U.S. Pat. No. 6,706,265, U.S.Pat. No. 5,585,097, U.S. Pat. No. 5,968,509, U.S. Pat. No. 5,932,448,U.S. Pat. No. 6,129,914, U.S. Pat. No. 7,381,803, U.S. Pat. No.5,834,597, and U.S. Pat. No. 7,862,813, which are incorporated byreference in their entirety. In some embodiments, the anti-FcRH5antibody binds an isoform c-specific region of the extracellular domainof FcRH5c. In some embodiments, the anti-FcRH5 antibodies binds Ig-likedomain 9 of FcRH5c.

Techniques for making multispecific antibodies include, but are notlimited to, recombinant co-expression of two immunoglobulin heavychain-light chain pairs having different specificities (see Milstein andCuello, Nature 305: 537 (1983)), WO 93/08829, and Traunecker et al.,EMBO J. 10: 3655 (1991)), and “knob-in-hole” engineering (see, e.g.,U.S. Pat. No. 5,731,168). Multi-specific antibodies may also be made byengineering electrostatic steering effects for making antibodyFc-heterodimeric molecules (WO 2009/089004A1); cross-linking two or moreantibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennanet al., Science, 229: 81 (1985)); using leucine zippers to producebi-specific antibodies (see, e.g., Kostelny et al., J. Immunol.,148(5):1547-1553 (1992)); using “diabody” technology for makingbispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl.Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (sFv)dimers (see, e.g. Gruber et al., J. Immunol., 152:5368 (1994)); andpreparing trispecific antibodies as described, e.g., in Tutt et al. J.Immunol. 147: 60 (1991).

Engineered antibodies with three or more functional antigen bindingsites, including “Octopus antibodies,” are also included herein (see,e.g. US 2006/0025576A1).

The antibody or fragment herein also includes a “Dual Acting FAb” or“DAF” comprising an antigen binding site that binds to FcRH5 as well asanother, different antigen (see, US 2008/0069820, for example).

According to a different approach, antibody variable domains with thedesired binding specificities (antibody-antigen combining sites) arefused to immunoglobulin constant domain sequences. Preferably, thefusion is with an Ig heavy chain constant domain, comprising at leastpart of the hinge, C_(H2), and C_(H3) regions. It is preferred to havethe first heavy-chain constant region (C_(H1)) containing the sitenecessary for light chain bonding, present in at least one of thefusions. DNAs encoding the immunoglobulin heavy chain fusions and, ifdesired, the immunoglobulin light chain, are inserted into separateexpression vectors, and are co-transfected into a suitable host cell.This provides for greater flexibility in adjusting the mutualproportions of the three polypeptide fragments in embodiments whenunequal ratios of the three polypeptide chains used in the constructionprovide the optimum yield of the desired bispecific antibody. It is,however, possible to insert the coding sequences for two or all threepolypeptide chains into a single expression vector when the expressionof at least two polypeptide chains in equal ratios results in highyields or when the ratios have no significant affect on the yield of thedesired chain combination.

In some embodiments, the bispecific antibodies are composed of a hybridimmunoglobulin heavy chain with a first binding specificity in one arm,and a hybrid immunoglobulin heavy chain-light chain pair (providing asecond binding specificity) in the other arm. It was found that thisasymmetric structure facilitates the separation of the desiredbispecific compound from unwanted immunoglobulin chain combinations, asthe presence of an immunoglobulin light chain in only one half of thebispecific molecule provides for a facile way of separation. Thisapproach is disclosed in WO 94/04690. For further details of generatingbispecific antibodies see, for example, Suresh et al., Methods inEnzymology 121:210 (1986).

According to another approach described in U.S. Pat. No. 5,731,168, theinterface between a pair of antibody molecules can be engineered tomaximize the percentage of heterodimers which are recovered fromrecombinant cell culture. The preferred interface comprises at least apart of the C_(H3) domain. In this method, one or more small amino acidside chains from the interface of the first antibody molecule arereplaced with larger side chains (e.g., tyrosine or tryptophan).Compensatory “cavities” of identical or similar size to the large sidechain(s) are created on the interface of the second antibody molecule byreplacing large amino acid side chains with smaller ones (e.g., alanineor threonine). This provides a mechanism for increasing the yield of theheterodimer over other unwanted end-products such as homodimers.Bispecific antibodies produced in accordance with this approach arereferred to herein as “protuberance-into-cavity” antibodies.

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage. Brennan et al.,Science 229:81 (1985) describe a procedure wherein intact antibodies areproteolytically cleaved to generate F(ab′)₂ fragments. These fragmentsare reduced in the presence of the dithiol complexing agent, sodiumarsenite, to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Fab′-SH fragments from E. coli can be directly recovered and chemicallycoupled to form bispecific antibodies. Shalaby et al., J. Exp. Med. 175:217-225 (1992) describe the production of a fully humanized bispecificantibody F(ab′)₂ molecule. Each Fab′ fragment was separately secretedfrom E. coli and subjected to directed chemical coupling in vitro toform the bispecific antibody. The bispecific antibody thus formed wasable to bind to cells overexpressing the ErbB2 receptor and normal humanT cells, as well as trigger the lytic activity of human cytotoxiclymphocytes against human breast tumor targets.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol. 148(5):1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., Proc. Natl. Acad.Sci. USA 90:6444-6448 (1993) has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise a VHconnected to a VL by a linker which is too short to allow pairingbetween the two domains on the same chain. Accordingly, the VH and VLdomains of one fragment are forced to pair with the complementary VL andV domains of another fragment, thereby forming two antigen-bindingsites. Another strategy for making bispecific antibody fragments by theuse of single-chain Fv (sFv) dimers has also been reported. See Gruberet al., J. Immunol., 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60(1991).

8. Antibody Variants

In certain embodiments, amino acid sequence variants of the antibodiesprovided herein are contemplated. For example, it may be desirable toimprove the binding affinity and/or other biological properties of theantibody Amino acid sequence variants of an antibody may be prepared byintroducing appropriate modifications into the nucleotide sequenceencoding the antibody, or by peptide synthesis. Such modificationsinclude, for example, deletions from, and/or insertions into and/orsubstitutions of residues within the amino acid sequences of theantibody. Any combination of deletion, insertion, and substitution canbe made to arrive at the final construct, provided that the finalconstruct possesses the desired characteristics, e.g., antigen-binding.

a) Substitution, Insertion, and Deletion Variants

In certain embodiments, antibody variants having one or more amino acidsubstitutions are provided. Sites of interest for substitutionalmutagenesis include the HVRs and FRs. Conservative substitutions areshown in Table 1 under the heading of “preferred substitutions.” Moresubstantial changes are provided in Table 1 under the heading of“exemplary substitutions,” and as further described below in referenceto amino acid side chain classes Amino acid substitutions may beintroduced into an antibody of interest and the products screened for adesired activity, e.g., retained/improved antigen binding, decreasedimmunogenicity, or improved ADCC or CDC.

TABLE 1 Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His;Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn;Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; ArgArg Ile (I) Leu; Val; Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine;Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe;Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr;Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Norleucine Leu

Amino acids may be grouped according to common side-chain properties:

(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

One type of substitutional variant involves substituting one or morehypervariable region residues of a parent antibody (e.g. a humanized orhuman antibody). Generally, the resulting variant(s) selected forfurther study will have modifications (e.g., improvements) in certainbiological properties (e.g., increased affinity, reduced immunogenicity)relative to the parent antibody and/or will have substantially retainedcertain biological properties of the parent antibody. An exemplarysubstitutional variant is an affinity matured antibody, which may beconveniently generated, e.g., using phage display-based affinitymaturation techniques such as those described herein. Briefly, one ormore HVR residues are mutated and the variant antibodies displayed onphage and screened for a particular biological activity (e.g. bindingaffinity).

Alterations (e.g., substitutions) may be made in HVRs, e.g., to improveantibody affinity. Such alterations may be made in HVR “hotspots,” i.e.,residues encoded by codons that undergo mutation at high frequencyduring the somatic maturation process (see, e.g., Chowdhury, MethodsMol. Biol. 207:179-196 (2008)), and/or SDRs (a-CDRs), with the resultingvariant VH or VL being tested for binding affinity. Affinity maturationby constructing and reselecting from secondary libraries has beendescribed, e.g., in Hoogenboom et al. in Methods in Molecular Biology178:1-37 (O'Brien et al., ed., Human Press, Totowa, N.J., (2001).) Insome embodiments of affinity maturation, diversity is introduced intothe variable genes chosen for maturation by any of a variety of methods(e.g., error-prone PCR, chain shuffling, or oligonucleotide-directedmutagenesis). A secondary library is then created. The library is thenscreened to identify any antibody variants with the desired affinity.Another method to introduce diversity involves HVR-directed approaches,in which several HVR residues (e.g., 4-6 residues at a time) arerandomized HVR residues involved in antigen binding may be specificallyidentified, e.g., using alanine scanning mutagenesis or modeling. CDR-H3and CDR-L3 in particular are often targeted.

In certain embodiments, substitutions, insertions, or deletions mayoccur within one or more HVRs so long as such alterations do notsubstantially reduce the ability of the antibody to bind antigen. Forexample, conservative alterations (e.g., conservative substitutions asprovided herein) that do not substantially reduce binding affinity maybe made in HVRs. Such alterations may be outside of HVR “hotspots” orSDRs. In certain embodiments of the variant VH and VL sequences providedabove, each HVR either is unaltered, or contains no more than one, twoor three amino acid substitutions.

A useful method for identification of residues or regions of an antibodythat may be targeted for mutagenesis is called “alanine scanningmutagenesis” as described by Cunningham and Wells (1989) Science,244:1081-1085. In this method, a residue or group of target residues(e.g., charged residues such as arg, asp, his, lys, and glu) areidentified and replaced by a neutral or negatively charged amino acid(e.g., alanine or polyalanine) to determine whether the interaction ofthe antibody with antigen is affected. Further substitutions may beintroduced at the amino acid locations demonstrating functionalsensitivity to the initial substitutions. Alternatively, oradditionally, a crystal structure of an antigen-antibody complex is usedto identify contact points between the antibody and antigen. Suchcontact residues and neighboring residues may be targeted or eliminatedas candidates for substitution. Variants may be screened to determinewhether they contain the desired properties.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue. Other insertionalvariants of the antibody molecule include the fusion to the N- orC-terminus of the antibody to an enzyme (e.g. for ADEPT) or apolypeptide which increases the serum half-life of the antibody.

b) Glycosylation Variants

In certain embodiments, an antibody provided herein is altered toincrease or decrease the extent to which the antibody is glycosylated.Addition or deletion of glycosylation sites to an antibody may beconveniently accomplished by altering the amino acid sequence such thatone or more glycosylation sites is created or removed.

Where the antibody comprises an Fc region, the carbohydrate attachedthereto may be altered. Native antibodies produced by mammalian cellstypically comprise a branched, biantennary oligosaccharide that isgenerally attached by an N-linkage to Asn297 of the CH2 domain of the Fcregion. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). Theoligosaccharide may include various carbohydrates, e.g., mannose,N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as afucose attached to a GlcNAc in the “stem” of the biantennaryoligosaccharide structure. In some embodiments, modifications of theoligosaccharide in an antibody provided herein may be made in order tocreate antibody variants with certain improved properties.

In one embodiment, antibody variants are provided having a carbohydratestructure that lacks fucose attached (directly or indirectly) to an Fcregion. For example, the amount of fucose in such antibody may be from1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%. The amountof fucose is determined by calculating the average amount of fucosewithin the sugar chain at Asn297, relative to the sum of allglycostructures attached to Asn 297 (e. g. complex, hybrid and highmannose structures) as measured by MALDI-TOF mass spectrometry, asdescribed in WO 2008/077546, for example. Asn297 refers to theasparagine residue located at about position 297 in the Fc region (Eunumbering of Fc region residues); however, Asn297 may also be locatedabout ±3 amino acids upstream or downstream of position 297, i.e.,between positions 294 and 300, due to minor sequence variations inantibodies. Such fucosylation variants may have improved ADCC function.See, e.g., US Patent Publication Nos. US 2003/0157108 (Presta, L.); US2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publicationsrelated to “defucosylated” or “fucose-deficient” antibody variantsinclude: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614;US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO2005/035586; WO 2005/035778; WO2005/053742; WO2002/031140; Okazaki etal. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech.Bioeng. 87: 614 (2004). Examples of cell lines capable of producingdefucosylated antibodies include Lec13 CHO cells deficient in proteinfucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986);US Pat Appl No US 2003/0157108 A1, Presta, L; and WO 2004/056312 A1,Adams et al., especially at Example 11), and knockout cell lines, suchas alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see,e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. etal., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003/085107).

Antibodies variants are further provided with bisected oligosaccharides,e.g., in which a biantennary oligosaccharide attached to the Fc regionof the antibody is bisected by GlcNAc. Such antibody variants may havereduced fucosylation and/or improved ADCC function. Examples of suchantibody variants are described, e.g., in WO 2003/011878 (Jean-Mairet etal.); U.S. Pat. No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umanaet al.). Antibody variants with at least one galactose residue in theoligosaccharide attached to the Fc region are also provided. Suchantibody variants may have improved CDC function. Such antibody variantsare described, e.g., in WO 1997/30087 (Patel et al.); WO 1998/58964(Raju, S.); and WO 1999/22764 (Raju, S.).

c) Fc Region Variants

In certain embodiments, one or more amino acid modifications may beintroduced into the Fc region of an antibody provided herein, therebygenerating an Fc region variant. The Fc region variant may comprise ahuman Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fcregion) comprising an amino acid modification (e.g. a substitution) atone or more amino acid positions.

In certain embodiments, the invention contemplates an antibody variantthat possesses some but not all effector functions, which make it adesirable candidate for applications in which the half-life of theantibody in vivo is important yet certain effector functions (such ascomplement and ADCC) are unnecessary or deleterious. In vitro and/or invivo cytotoxicity assays can be conducted to confirm thereduction/depletion of CDC and/or ADCC activities. For example, Fcreceptor (FcR) binding assays can be conducted to ensure that theantibody lacks FcγR binding (hence likely lacking ADCC activity), butretains FcRn binding ability. The primary cells for mediating ADCC, NKcells, express Fc(RIII only, whereas monocytes express Fc(RI, Fc(RII andFc(RIII. FcR expression on hematopoietic cells is summarized in Table 3on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991).Non-limiting examples of in vitro assays to assess ADCC activity of amolecule of interest is described in U.S. Pat. No. 5,500,362 (see, e.g.Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) andHellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985);U.S. Pat. No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med.166:1351-1361 (1987)). Alternatively, non-radioactive assays methods maybe employed (see, for example, ACTI™ non-radioactive cytotoxicity assayfor flow cytometry (CellTechnology, Inc. Mountain View, Calif.; andCytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, Wis.).Useful effector cells for such assays include peripheral bloodmononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively,or additionally, ADCC activity of the molecule of interest may beassessed in vivo, e.g., in an animal model such as that disclosed inClynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q bindingassays may also be carried out to confirm that the antibody is unable tobind C1q and hence lacks CDC activity. See, e.g., C1q and C3c bindingELISA in WO 2006/029879 and WO 2005/100402. To assess complementactivation, a CDC assay may be performed (see, for example,Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, M. S.et al., Blood 101:1045-1052 (2003); and Cragg, M. S. and M. J. Glennie,Blood 103:2738-2743 (2004)). FcRn binding and in vivoclearance/half-life determinations can also be performed using methodsknown in the art (see, e.g., Petkova, S. B. et al., Int'l. Immunol.18(12):1759-1769 (2006)).

Antibodies with reduced effector function include those withsubstitution of one or more of Fc region residues 238, 265, 269, 270,297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fcmutants with substitutions at two or more of amino acid positions 265,269, 270, 297 and 327, including the so-called “DANA” Fc mutant withsubstitution of residues 265 and 297 to alanine (U.S. Pat. No.7,332,581).

Certain antibody variants with improved or diminished binding to FcRsare described. (See, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312, andShields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).)

In certain embodiments, an antibody variant comprises an Fc region withone or more amino acid substitutions which improve ADCC, e.g.,substitutions at positions 298, 333, and/or 334 of the Fc region (EUnumbering of residues).

In some embodiments, alterations are made in the Fc region that resultin altered (i.e., either improved or diminished) C1q binding and/orComplement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat.No. 6,194,551, WO 99/51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

Antibodies with increased half-lives and improved binding to theneonatal Fc receptor (FcRn), which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) andKim et al., J. Immunol. 24:249 (1994)), are described inUS2005/0014934A1 (Hinton et al.). Those antibodies comprise an Fc regionwith one or more substitutions therein which improve binding of the Fcregion to FcRn. Such Fc variants include those with substitutions at oneor more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307,311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434,e.g., substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826).

See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Pat. No.5,648,260; U.S. Pat. No. 5,624,821; and WO 94/29351 concerning otherexamples of Fc region variants.

d) Cysteine Engineered Antibody Variants

In certain embodiments, it may be desirable to create cysteineengineered antibodies, e.g., “thioMAbs,” in which one or more residuesof an antibody are substituted with cysteine residues. In particularembodiments, the substituted residues occur at accessible sites of theantibody. By substituting those residues with cysteine, reactive thiolgroups are thereby positioned at accessible sites of the antibody andmay be used to conjugate the antibody to other moieties, such as drugmoieties or linker-drug moieties, to create an immunoconjugate, asdescribed further herein. In certain embodiments, any one or more of thefollowing residues may be substituted with cysteine: V205 (Kabatnumbering) of the light chain; A118 (EU numbering) of the heavy chain;and S400 (EU numbering) of the heavy chain Fc region. Cysteineengineered antibodies may be generated as described, e.g., in U.S. Pat.No. 7,521,541.

e) Antibody Derivatives

In certain embodiments, an antibody provided herein may be furthermodified to contain additional nonproteinaceous moieties that are knownin the art and readily available. The moieties suitable forderivatization of the antibody include but are not limited to watersoluble polymers. Non-limiting examples of water soluble polymersinclude, but are not limited to, polyethylene glycol (PEG), copolymersof ethylene glycol/propylene glycol, carboxymethylcellulose, dextran,polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane,poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids(either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propropylene glycol homopolymers,prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylatedpolyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.Polyethylene glycol propionaldehyde may have advantages in manufacturingdue to its stability in water. The polymer may be of any molecularweight, and may be branched or unbranched. The number of polymersattached to the antibody may vary, and if more than one polymer areattached, they can be the same or different molecules. In general, thenumber and/or type of polymers used for derivatization can be determinedbased on considerations including, but not limited to, the particularproperties or functions of the antibody to be improved, whether theantibody derivative will be used in a therapy under defined conditions,etc.

In another embodiment, conjugates of an antibody and nonproteinaceousmoiety that may be selectively heated by exposure to radiation areprovided. In one embodiment, the nonproteinaceous moiety is a carbonnanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605(2005)). The radiation may be of any wavelength, and includes, but isnot limited to, wavelengths that do not harm ordinary cells, but whichheat the nonproteinaceous moiety to a temperature at which cellsproximal to the antibody-nonproteinaceous moiety are killed.

B. Recombinant Methods and Compositions

Antibodies may be produced using recombinant methods and compositions,e.g., as described in U.S. Pat. No. 4,816,567. In one embodiment,isolated nucleic acid encoding an anti-FcRH5 antibody described hereinis provided. Such nucleic acid may encode an amino acid sequencecomprising the VL and/or an amino acid sequence comprising the VH of theantibody (e.g., the light and/or heavy chains of the antibody). In afurther embodiment, one or more vectors (e.g., expression vectors)comprising such nucleic acid are provided. In a further embodiment, ahost cell comprising such nucleic acid is provided. In one suchembodiment, a host cell comprises (e.g., has been transformed with): (1)a vector comprising a nucleic acid that encodes an amino acid sequencecomprising the VL of the antibody and an amino acid sequence comprisingthe VH of the antibody, or (2) a first vector comprising a nucleic acidthat encodes an amino acid sequence comprising the VL of the antibodyand a second vector comprising a nucleic acid that encodes an amino acidsequence comprising the VH of the antibody. In one embodiment, the hostcell is eukaryotic, e.g. a Chinese Hamster Ovary (CHO) cell or lymphoidcell (e.g., Y0, NS0, Sp20 cell). In one embodiment, a method of makingan anti-FcRH5 antibody is provided, wherein the method comprisesculturing a host cell comprising a nucleic acid encoding the antibody,as provided above, under conditions suitable for expression of theantibody, and optionally recovering the antibody from the host cell (orhost cell culture medium).

For recombinant production of an anti-FcRH5 antibody, nucleic acidencoding an antibody, e.g., as described above, is isolated and insertedinto one or more vectors for further cloning and/or expression in a hostcell. Such nucleic acid may be readily isolated and sequenced usingconventional procedures (e.g., by using oligonucleotide probes that arecapable of binding specifically to genes encoding the heavy and lightchains of the antibody).

Suitable host cells for cloning or expression of antibody-encodingvectors include prokaryotic or eukaryotic cells described herein. Forexample, antibodies may be produced in bacteria, in particular whenglycosylation and Fc effector function are not needed. For expression ofantibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat.Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods inMolecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa,N.J., 2003), pp. 245-254, describing expression of antibody fragments inE. coli.) After expression, the antibody may be isolated from thebacterial cell paste in a soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors, including fungi and yeast strains whoseglycosylation pathways have been “humanized,” resulting in theproduction of an antibody with a partially or fully human glycosylationpattern. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li etal., Nat. Biotech. 24:210-215 (2006).

Suitable host cells for the expression of glycosylated antibody are alsoderived from multicellular organisms (invertebrates and vertebrates).Examples of invertebrate cells include plant and insect cells. Numerousbaculoviral strains have been identified which may be used inconjunction with insect cells, particularly for transfection ofSpodoptera frugiperda cells.

Plant cell cultures can also be utilized as hosts. See, e.g., U.S. Pat.Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429(describing PLANTIBODIES™ technology for producing antibodies intransgenic plants).

Vertebrate cells may also be used as hosts. For example, mammalian celllines that are adapted to grow in suspension may be useful. Otherexamples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7); human embryonic kidney line (293 or 293cells as described, e.g., in Graham et al., J. Gen Virol. 36:59 (1977));baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells asdescribed, e.g., in Mather, Biol. Reprod. 23:243-251 (1980)); monkeykidney cells (CV1); African green monkey kidney cells (VERO-76); humancervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo ratliver cells (BRL 3A); human lung cells (W138); human liver cells (HepG2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., inMather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; andFS4 cells. Other useful mammalian host cell lines include Chinesehamster ovary (CHO) cells, including DHFR⁻ CHO cells (Urlaub et al.,Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines suchas Y0, NS0 and Sp2/0. For a review of certain mammalian host cell linessuitable for antibody production, see, e.g., Yazaki and Wu, Methods inMolecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa,N.J.), pp. 255-268 (2003).

C. Assays

Anti-FcRH5 antibodies provided herein may be identified, screened for,or characterized for their physical/chemical properties and/orbiological activities by various assays known in the art.

In one aspect, an antibody provided herein may be tested for its antigenbinding activity, e.g., by known methods such as ELISA, BIACore®, FACS,or Western blot.

In another aspect, competition assays may be used to identify anantibody that competes with any of the antibodies described herein forbinding to FcRH5. In certain embodiments, such a competing antibodybinds to the same epitope (e.g., a linear or a conformational epitope)that is bound by an antibody described herein. Detailed exemplarymethods for mapping an epitope to which an antibody binds are providedin Morris (1996) “Epitope Mapping Protocols,” in Methods in MolecularBiology vol. 66 (Humana Press, Totowa, N.J.).

In an exemplary competition assay, immobilized FcRH5 is incubated in asolution comprising a first labeled antibody that binds to FcRH5 (e.g.,any of the antibodies described herein) and a second unlabeled antibodythat is being tested for its ability to compete with the first antibodyfor binding to FcRH5. The second antibody may be present in a hybridomasupernatant. As a control, immobilized FcRH5 is incubated in a solutioncomprising the first labeled antibody but not the second unlabeledantibody. After incubation under conditions permissive for binding ofthe first antibody to FcRH5, excess unbound antibody is removed, and theamount of label associated with immobilized FcRH5 is measured. If theamount of label associated with immobilized FcRH5 is substantiallyreduced in the test sample relative to the control sample, then thatindicates that the second antibody is competing with the first antibodyfor binding to FcRH5. See Harlow and Lane (1988) Antibodies: ALaboratory Manual ch. 14 (Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.). In some embodiments, the FcRH5 is FcRH5c. In someembodiments, the anti-FcRH5 antibody binds an isoform c-specific regionof the extracellular domain of FcRH5c. In some embodiments, theanti-FcRH5 antibodies binds Ig-like domain 9 of FcRH5c.

D. Immunoconjugates

Also provided herein are immunoconjugates comprising an anti-FcRH5antibody herein conjugated to one or more cytotoxic agents, such aschemotherapeutic agents or drugs, growth inhibitory agents, toxins(e.g., protein toxins, enzymatically active toxins of bacterial, fungal,plant, or animal origin, or fragments thereof), or radioactive isotopes(i.e., a radioconjugate). In some embodiments, the anti-FcRH5 antibodybinds an isoform c-specific region of the extracellular domain ofFcRH5c. In some embodiments, the anti-FcRH5 antibodies binds Ig-likedomain 9 of FcRH5c.

Immunoconjugates allow for the targeted delivery of a drug moiety to atumor, and, in some embodiments intracellular accumulation therein,where systemic administration of unconjugated drugs may result inunacceptable levels of toxicity to normal cells (Polakis P. (2005)Current Opinion in Pharmacology 5:382-387).

Antibody-drug conjugates (ADC) are targeted chemotherapeutic moleculeswhich combine properties of both antibodies and cytotoxic drugs bytargeting potent cytotoxic drugs to antigen-expressing tumor cells(Teicher, B. A. (2009) Current Cancer Drug Targets 9:982-1004), therebyenhancing the therapeutic index by maximizing efficacy and minimizingoff-target toxicity (Carter, P. J. and Senter P. D. (2008) The CancerJour. 14(3):154-169; Chari, R. V. (2008) Acc. Chem. Res. 41:98-107.

The ADC compounds provided herein include those with anticanceractivity. In some embodiments, the ADC compounds include an antibodyconjugated, i.e. covalently attached, to the drug moiety. In someembodiments, the antibody is covalently attached to the drug moietythrough a linker. The antibody-drug conjugates (ADC) provided hereinselectively deliver an effective dose of a drug to tumor tissue wherebygreater selectivity, i.e. a lower efficacious dose, may be achievedwhile increasing the therapeutic index (“therapeutic window”).

The drug moiety (D) of the antibody-drug conjugates (ADC) may includeany compound, moiety or group that has a cytotoxic or cytostatic effect.Drug moieties may impart their cytotoxic and cytostatic effects bymechanisms including but not limited to tubulin binding, DNA binding orintercalation, and inhibition of RNA polymerase, protein synthesis,and/or topoisomerase. Exemplary drug moieties include, but are notlimited to, a maytansinoid, dolastatin, auristatin, calicheamicin,pyrrolobenzodiazepine (PBD), nemorubicin and its derivatives,PNU-159682, anthracycline, duocarmycin, vinca alkaloid, taxane,trichothecene, CC1065, camptothecin, elinafide, and stereoisomers,isosteres, analogs, and derivatives thereof that have cytotoxicactivity. Nonlimiting examples of such immunoconjugates are discussed infurther detail below.

1. Exemplary Antibody-Drug Conjugates

An exemplary embodiment of an antibody-drug conjugate (ADC) compoundcomprises an antibody (Ab) which targets a tumor cell, a drug moiety(D), and a linker moiety (L) that attaches Ab to D. In some embodiments,the antibody is attached to the linker moiety (L) through one or moreamino acid residues, such as lysine and/or cysteine.

An exemplary ADC has Formula I:

Ab-(L-D)_(p)  I

where p is 1 to about 20. In some embodiments, the number of drugmoieties that can be conjugated to an antibody is limited by the numberof free cysteine residues. In some embodiments, free cysteine residuesare introduced into the antibody amino acid sequence by the methodsdescribed herein. Exemplary ADC of Formula I include, but are notlimited to, antibodies that have 1, 2, 3, or 4 engineered cysteine aminoacids (Lyon, R. et al (2012) Methods in Enzym. 502:123-138). In someembodiments, one or more free cysteine residues are already present inan antibody, without the use of engineering, in which case the existingfree cysteine residues may be used to conjugate the antibody to a drug.In some embodiments, an antibody is exposed to reducing conditions priorto conjugation of the antibody in order to generate one or more freecysteine residues. In some embodiments, the anti-FcRH5 antibody binds anisoform c-specific region of the extracellular domain of FcRH5c. In someembodiments, the anti-FcRH5 antibodies binds Ig-like domain 9 of FcRH5c.

a) Exemplary Linkers

A “Linker” (L) is a bifunctional or multifunctional moiety that can beused to link one or more drug moieties (D) to an antibody (Ab) to forman antibody-drug conjugate (ADC) of Formula I. In some embodiments,antibody-drug conjugates (ADC) can be prepared using a Linker havingreactive functionalities for covalently attaching to the drug and to theantibody. For example, in some embodiments, a cysteine thiol of anantibody (Ab) can form a bond with a reactive functional group of alinker or a drug-linker intermediate to make an ADC.

In one aspect, a linker has a functionality that is capable of reactingwith a free cysteine present on an antibody to form a covalent bond.Nonlimiting exemplary such reactive functionalities include maleimide,haloacetamides, α-haloacetyl, activated esters such as succinimideesters, 4-nitrophenyl esters, pentafluorophenyl esters,tetrafluorophenyl esters, anhydrides, acid chlorides, sulfonylchlorides, isocyanates, and isothiocyanates. See, e.g., the conjugationmethod at page 766 of Klussman, et al (2004), Bioconjugate Chemistry15(4):765-773, and the Examples herein.

In some embodiments, a linker has a functionality that is capable ofreacting with an electrophilic group present on an antibody. Exemplarysuch electrophilic groups include, but are not limited to, aldehyde andketone carbonyl groups. In some embodiments, a heteroatom of thereactive functionality of the linker can react with an electrophilicgroup on an antibody and form a covalent bond to an antibody unit.Nonlimiting exemplary such reactive functionalities include, but are notlimited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone,hydrazine carboxylate, and arylhydrazide.

A linker may comprise one or more linker components. Exemplary linkercomponents include 6-maleimidocaproyl (“MC”), maleimidopropanoyl (“MP”),valine-citrulline (“val-cit” or “vc”), alanine-phenylalanine(“ala-phe”), p-aminobenzyloxycarbonyl (a “PAB”), N-Succinimidyl4-(2-pyridylthio) pentanoate (“SPP”), and 4-(N-maleimidomethyl)cyclohexane-1 carboxylate (“MCC”). Various linker components are knownin the art, some of which are described below.

A linker may be a “cleavable linker,” facilitating release of a drug.Nonlimiting exemplary cleavable linkers include acid-labile linkers(e.g., comprising hydrazone), protease-sensitive (e.g.,peptidase-sensitive) linkers, photolabile linkers, ordisulfide-containing linkers (Chari et al., Cancer Research 52:127-131(1992); U.S. Pat. No. 5,208,020).

In certain embodiments, a linker has the following Formula II:

-A_(a)-W_(w)—Y_(y)-  II

wherein A is a “stretcher unit”, and a is an integer from 0 to 1; W isan “amino acid unit”, and w is an integer from 0 to 12; Y is a “spacerunit”, and y is 0, 1, or 2. An ADC comprising the linker of Formula IIhas the Formula I(A): Ab-(A_(a)-W_(w)—Y_(y)-D)p, wherein Ab, D, and pare defined as above for Formula I. Exemplary embodiments of suchlinkers are described in U.S. Pat. No. 7,498,298, which is expresslyincorporated herein by reference.

In some embodiments, a linker component comprises a “stretcher unit” (A)that links an antibody to another linker component or to a drug moiety.Nonlimiting exemplary stretcher units are shown below (wherein the wavyline indicates sites of covalent attachment to an antibody, drug, oradditional linker components):

In some embodiments, a linker component comprises an “amino acid unit”(W). In some such embodiments, the amino acid unit allows for cleavageof the linker by a protease, thereby facilitating release of the drugfrom the immunoconjugate upon exposure to intracellular proteases, suchas lysosomal enzymes (Doronina et al. (2003) Nat. Biotechnol.21:778-784). Exemplary amino acid units include, but are not limited to,dipeptides, tripeptides, tetrapeptides, and pentapeptides. Exemplarydipeptides include, but are not limited to, valine-citrulline (vc orval-cit), alanine-phenylalanine (af or ala-phe); phenylalanine-lysine(fk or phe-lys); phenylalanine-homolysine (phe-homolys); andN-methyl-valine-citrulline (Me-val-cit). Exemplary tripeptides include,but are not limited to, glycine-valine-citrulline (gly-val-cit) andglycine-glycine-glycine (gly-gly-gly). An amino acid unit may compriseamino acid residues that occur naturally and/or minor amino acids and/ornon-naturally occurring amino acid analogs, such as citrulline Aminoacid units can be designed and optimized for enzymatic cleavage by aparticular enzyme, for example, a tumor-associated protease, cathepsinB, C and D, or a plasmin protease.

Typically, peptide-type linkers can be prepared by forming a peptidebond between two or more amino acids and/or peptide fragments. Suchpeptide bonds can be prepared, for example, according to a liquid phasesynthesis method (e.g., E. Schröder and K. Lübke (1965) “The Peptides”,volume 1, pp 76-136, Academic Press).

In some embodiments, a linker component comprises a “spacer unit” (Y)that links the antibody to a drug moiety, either directly or through astretcher unit and/or an amino acid unit. A spacer unit may be“self-immolative” or a “non-self-immolative.” A “non-self-immolative”spacer unit is one in which part or all of the spacer unit remains boundto the drug moiety upon cleavage of the ADC. Examples ofnon-self-immolative spacer units include, but are not limited to, aglycine spacer unit and a glycine-glycine spacer unit. In someembodiments, enzymatic cleavage of an ADC containing a glycine-glycinespacer unit by a tumor-cell associated protease results in release of aglycine-glycine-drug moiety from the remainder of the ADC. In some suchembodiments, the glycine-glycine-drug moiety is subjected to ahydrolysis step in the tumor cell, thus cleaving the glycine-glycinespacer unit from the drug moiety.

A “self-immolative” spacer unit allows for release of the drug moiety.In certain embodiments, a spacer unit of a linker comprises ap-aminobenzyl unit. In some such embodiments, a p-aminobenzyl alcohol isattached to an amino acid unit via an amide bond, and a carbamate,methylcarbamate, or carbonate is made between the benzyl alcohol and thedrug (Hamann et al. (2005) Expert Opin. Ther. Patents (2005)15:1087-1103). In some embodiments, the spacer unit comprisesp-aminobenzyloxycarbonyl (PAB). In some embodiments, an ADC comprising aself-immolative linker has the structure:

wherein Q is —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -halogen, -nitro, or-cyano; m is an integer ranging from 0 to 4; X may be one or moreadditional spacer units or may be absent; and p ranges from 1 to about20. In some embodiments, p ranges from 1 to 10, 1 to 7, 1 to 5, or 1 to4. Nonlimiting exemplary X spacer units include:

wherein R₁ and R₂ are independently selected from H and C₁-C₆ alkyl. Insome embodiments, R1 and R2 are each —CH₃.

Other examples of self-immolative spacers include, but are not limitedto, aromatic compounds that are electronically similar to the PAB group,such as 2-aminoimidazol-5-methanol derivatives (U.S. Pat. No. 7,375,078;Hay et al. (1999) Bioorg. Med. Chem. Lett. 9:2237) and ortho- orpara-aminobenzylacetals. In some embodiments, spacers can be used thatundergo cyclization upon amide bond hydrolysis, such as substituted andunsubstituted 4-aminobutyric acid amides (Rodrigues et al (1995)Chemistry Biology 2:223), appropriately substituted bicyclo[2.2.1] andbicyclo[2.2.2] ring systems (Storm et al (1972) J. Amer. Chem. Soc.94:5815) and 2-aminophenylpropionic acid amides (Amsberry, et al (1990)J. Org. Chem. 55:5867). Linkage of a drug to the α-carbon of a glycineresidue is another example of a self-immolative spacer that may beuseful in ADC (Kingsbury et al (1984) J. Med. Chem. 27:1447).

In some embodiments, linker L may be a dendritic type linker forcovalent attachment of more than one drug moiety to an antibody througha branching, multifunctional linker moiety (Sun et al (2002) Bioorganic& Medicinal Chemistry Letters 12:2213-2215; Sun et al (2003) Bioorganic& Medicinal Chemistry 11:1761-1768). Dendritic linkers can increase themolar ratio of drug to antibody, i.e. loading, which is related to thepotency of the ADC. Thus, where an antibody bears only one reactivecysteine thiol group, a multitude of drug moieties may be attachedthrough a dendritic linker.

Nonlimiting exemplary linkers are shown below in the context of an ADCof Formula I:

wherein R₁ and R₂ are independently selected from H and C₁-C₆ alkyl. Insome embodiments, R1 and R2 are each —CH₃.

wherein n is 0 to 12. In some embodiments, n is 2 to 10. In someembodiments, n is 4 to 8.

Further nonlimiting exemplary ADCs include the structures:

where X is:

Y is:

each R is independently H or C₁-C₆ alkyl; and n is 1 to 12.

In some embodiments, a linker is substituted with groups that modulatesolubility and/or reactivity. As a nonlimiting example, a chargedsubstituent such as sulfonate (—SO₃ ⁻) or ammonium may increase watersolubility of the linker reagent and facilitate the coupling reaction ofthe linker reagent with the antibody and/or the drug moiety, orfacilitate the coupling reaction of Ab-L (antibody-linker intermediate)with D, or D-L (drug-linker intermediate) with Ab, depending on thesynthetic route employed to prepare the ADC. In some embodiments, aportion of the linker is coupled to the antibody and a portion of thelinker is coupled to the drug, and then the Ab-(linker portion)^(a) iscoupled to drug-(linker portion)^(b) to form the ADC of Formula I.

The compounds provided herein expressly contemplate, but are not limitedto, ADC prepared with the following linker reagents:bis-maleimido-trioxyethylene glycol (BMPEO),N-(β-maleimidopropyloxy)-N-hydroxy succinimide ester (BMPS),N-(ε-maleimidocaproyloxy) succinimide ester (EMCS),N-[γ-maleimidobutyryloxy]succinimide ester (GMBS),1,6-hexane-bis-vinylsulfone (HBVS), succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxy-(6-amidocaproate) (LC-SMCC),m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS),4-(4-N-Maleimidophenyl)butyric acid hydrazide (MPBH), succinimidyl3-(bromoacetamido)propionate (SBAP), succinimidyl iodoacetate (SIA),succinimidyl (4-iodoacetyl)aminobenzoate (SLAB),N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),N-succinimidyl-4-(2-pyridylthio)pentanoate (SPP), succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), succinimidyl4-(p-maleimidophenyl)butyrate (SMPB), succinimidyl6-[(beta-maleimidopropionamido)hexanoate] (SMPH), iminothiolane (IT),sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC,and sulfo-SMPB, and succinimidyl-(4-vinylsulfone)benzoate (SVSB), andincluding bis-maleimide reagents: dithiobismaleimidoethane (DTME),1,4-Bismaleimidobutane (BMB), 1,4 Bismaleimidyl-2,3-dihydroxybutane(BMDB), bismaleimidohexane (BMH), bismaleimidoethane (BMOE), BM(PEG)₂(shown below), and BM(PEG)₃ (shown below); bifunctional derivatives ofimidoesters (such as dimethyl adipimidate HCl), active esters (such asdisuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azidocompounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazoniumderivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),diisocyanates (such as toluene 2,6-diisocyanate), and bis-activefluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). In someembodiments, bis-maleimide reagents allow the attachment of the thiolgroup of a cysteine in the antibody to a thiol-containing drug moiety,linker, or linker-drug intermediate. Other functional groups that arereactive with thiol groups include, but are not limited to,iodoacetamide, bromoacetamide, vinyl pyridine, disulfide, pyridyldisulfide, isocyanate, and isothiocyanate.

Certain useful linker reagents can be obtained from various commercialsources, such as Pierce Biotechnology, Inc. (Rockford, Ill.), MolecularBiosciences Inc. (Boulder, Colo.), or synthesized in accordance withprocedures described in the art; for example, in Toki et al (2002) J.Org. Chem. 67:1866-1872; Dubowchik, et al. (1997) Tetrahedron Letters,38:5257-60; Walker, M. A. (1995) J. Org. Chem. 60:5352-5355; Frisch etal (1996) Bioconjugate Chem. 7:180-186; U.S. Pat. No. 6,214,345; WO02/088172; US 2003130189; US2003096743; WO 03/026577; WO 03/043583; andWO 04/032828.

Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See, e.g., WO94/11026.

b) Drug Moieties

(1) Maytansine and Maytansinoids

In some embodiments, an immunoconjugate comprises an antibody conjugatedto one or more maytansinoid molecules. Maytansinoids are derivatives ofmaytansine, and are mitototic inhibitors which act by inhibiting tubulinpolymerization. Maytansine was first isolated from the east Africanshrub Maytenus serrata (U.S. Pat. No. 3,896,111). Subsequently, it wasdiscovered that certain microbes also produce maytansinoids, such asmaytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042).Synthetic maytansinoids are disclosed, for example, in U.S. Pat. Nos.4,137,230; 4,248,870; 4,256,746; 4,260,608; 4,265,814; 4,294,757;4,307,016; 4,308,268; 4,308,269; 4,309,428; 4,313,946; 4,315,929;4,317,821; 4,322,348; 4,331,598; 4,361,650; 4,364,866; 4,424,219;4,450,254; 4,362,663; and 4,371,533.

Maytansinoid drug moieties are attractive drug moieties in antibody-drugconjugates because they are: (i) relatively accessible to prepare byfermentation or chemical modification or derivatization of fermentationproducts, (ii) amenable to derivatization with functional groupssuitable for conjugation through non-disulfide linkers to antibodies,(iii) stable in plasma, and (iv) effective against a variety of tumorcell lines.

Certain maytansinoids suitable for use as maytansinoid drug moieties areknown in the art and can be isolated from natural sources according toknown methods or produced using genetic engineering techniques (see,e.g., Yu et al (2002) PNAS 99:7968-7973). Maytansinoids may also beprepared synthetically according to known methods.

Maytansinoid drug moieties include, but are not limited to, those havinga modified aromatic ring, such as: C-19-dechloro (U.S. Pat. No.4,256,746) (prepared, for example, by lithium aluminum hydride reductionof ansamytocin P2); C-20-hydroxy (or C-20-demethyl) +/−C-19-dechloro(U.S. Pat. Nos. 4,361,650 and 4,307,016) (prepared, for example, bydemethylation using Streptomyces or Actinomyces or dechlorination usingLAH); and C-20-demethoxy, C-20-acyloxy (—OCOR), +/−dechloro (U.S. Pat.No. 4,294,757) (prepared, for example, by acylation using acylchlorides), and those having modifications at other positions of thearomatic ring.

Maytansinoid drug moieties also include those having modifications suchas: C-9-SH (U.S. Pat. No. 4,424,219) (prepared, for example, by thereaction of maytansinol with H₂S or P₂S₅);C-14-alkoxymethyl(demethoxy/CH₂OR)(U.S. Pat. No. 4,331,598);C-14-hydroxymethyl or acyloxymethyl (CH₂OH or CH₂OAc) (U.S. Pat. No.4,450,254) (prepared, for example, from Nocardia); C-15-hydroxy/acyloxy(U.S. Pat. No. 4,364,866) (prepared, for example, by the conversion ofmaytansinol by Streptomyces); C-15-methoxy (U.S. Pat. Nos. 4,313,946 and4,315,929) (for example, isolated from Trewia nudlflora);C-18-N-demethyl (U.S. Pat. Nos. 4,362,663 and 4,322,348) (prepared, forexample, by the demethylation of maytansinol by Streptomyces); and4,5-deoxy (U.S. Pat. No. 4,371,533) (prepared, for example, by thetitanium trichloride/LAH reduction of maytansinol).

Many positions on maytansinoid compounds are useful as the linkageposition. For example, an ester linkage may be formed by reaction with ahydroxyl group using conventional coupling techniques. In someembodiments, the reaction may occur at the C-3 position having ahydroxyl group, the C-14 position modified with hydroxymethyl, the C-15position modified with a hydroxyl group, and the C-20 position having ahydroxyl group. In some embodiments, the linkage is formed at the C-3position of maytansinol or a maytansinol analogue.

Maytansinoid drug moieties include those having the structure:

where the wavy line indicates the covalent attachment of the sulfur atomof the maytansinoid drug moiety to a linker of an ADC. Each R mayindependently be H or a C₁-C₆ alkyl. The alkylene chain attaching theamide group to the sulfur atom may be methanyl, ethanyl, or propyl,i.e., m is 1, 2, or 3 (U.S. Pat. No. 633,410; U.S. Pat. No. 5,208,020;Chari et al (1992) Cancer Res. 52:127-131; Liu et al (1996) Proc. Natl.Acad. Sci USA 93:8618-8623).

All stereoisomers of the maytansinoid drug moiety are contemplated forthe ADC provided herein, i.e. any combination of R and S configurationsat the chiral carbons (U.S. Pat. No. 7,276,497; U.S. Pat. No. 6,913,748;U.S. Pat. No. 6,441,163; U.S. Pat. No. 633,410 (RE39151); U.S. Pat. No.5,208,020; Widdison et al (2006) J. Med. Chem. 49:4392-4408, which areincorporated by reference in their entirety). In some embodiments, themaytansinoid drug moiety has the following stereochemistry:

Exemplary embodiments of maytansinoid drug moieties include, but are notlimited to, DM1; DM3; and DM4, having the structures:

wherein the wavy line indicates the covalent attachment of the sulfuratom of the drug to a linker (L) of an antibody-drug conjugate.

Other exemplary maytansinoid antibody-drug conjugates have the followingstructures and abbreviations (wherein Ab is antibody and p is 1 to about20. In some embodiments, p is 1 to 10, p is 1 to 7, p is 1 to 5, or p is1 to 4):

Exemplary antibody-drug conjugates where DM1 is linked through a BMPEOlinker to a thiol group of the antibody have the structure andabbreviation:

where Ab is antibody; n is 0, 1, or 2; and p is 1 to about 20. In someembodiments, p is 1 to 10, p is 1 to 7, p is 1 to 5, or p is 1 to 4.

Immunoconjugates containing maytansinoids, methods of making the same,and their therapeutic use are disclosed, for example, in U.S. Pat. Nos.5,208,020 and 5,416,064; US 2005/0276812 A1; and European Patent EP 0425 235 B1, the disclosures of which are hereby expressly incorporatedby reference. See also Liu et al. Proc. Natl. Acad. Sci. USA93:8618-8623 (1996); and Chari et al. Cancer Research 52:127-131 (1992).

In some embodiments, antibody-maytansinoid conjugates may be prepared bychemically linking an antibody to a maytansinoid molecule withoutsignificantly diminishing the biological activity of either the antibodyor the maytansinoid molecule. See, e.g., U.S. Pat. No. 5,208,020 (thedisclosure of which is hereby expressly incorporated by reference). Insome embodiments, ADC with an average of 3-4 maytansinoid moleculesconjugated per antibody molecule has shown efficacy in enhancingcytotoxicity of target cells without negatively affecting the functionor solubility of the antibody. In some instances, even one molecule oftoxin/antibody is expected to enhance cytotoxicity over the use of nakedantibody.

Linking groups for making antibody-maytansinoid conjugates include, forexample, those described herein and those disclosed in U.S. Pat. No.5,208,020; EP Patent 0 425 235 B1; Chari et al. Cancer Research52:127-131 (1992); US 2005/0276812 A1; and US 2005/016993 A1, thedisclosures of which are hereby expressly incorporated by reference.

(2) Auristatins and Dolastatins

Drug moieties include dolastatins, auristatins, and analogs andderivatives thereof (U.S. Pat. No. 5,635,483; U.S. Pat. No. 5,780,588;U.S. Pat. No. 5,767,237; U.S. Pat. No. 6,124,431). Auristatins arederivatives of the marine mollusk compound dolastatin-10. While notintending to be bound by any particular theory, dolastatins andauristatins have been shown to interfere with microtubule dynamics, GTPhydrolysis, and nuclear and cellular division (Woyke et al (2001)Antimicrob. Agents and Chemother. 45(12):3580-3584) and have anticancer(U.S. Pat. No. 5,663,149) and antifungal activity (Pettit et al (1998)Antimicrob. Agents Chemother. 42:2961-2965). The dolastatin/auristatindrug moiety may be attached to the antibody through the N (amino)terminus or the C (carboxyl) terminus of the peptidic drug moiety (WO02/088172; Doronina et al (2003) Nature Biotechnology 21(7):778-784;Francisco et al (2003) Blood 102(4):1458-1465).

Exemplary auristatin embodiments include the N-terminus linkedmonomethylauristatin drug moieties D_(E) and D_(F), disclosed in U.S.Pat. No. 7,498,298 and U.S. Pat. No. 7,659,241, the disclosures of whichare expressly incorporated by reference in their entirety:

wherein the wavy line of D_(E) and D_(F) indicates the covalentattachment site to an antibody or antibody-linker component, andindependently at each location:R² is selected from H and C₁-C₈ alkyl;R³ is selected from H, C₁-C₈ alkyl, C₃-C₈ carbocycle, aryl, C₁-C₈alkyl-aryl, C₁-C₈ alkyl-(C₃-C₈ carbocycle), C₃-C₈ heterocycle and C₁-C₈alkyl-(C₃-C₈ heterocycle);R⁴ is selected from H, C₁-C₈ alkyl, C₃-C₈ carbocycle, aryl, C₁-C₈alkyl-aryl, C₁-C₈ alkyl-(C₃-C₈ carbocycle), C₃-C₈ heterocycle and C₁-C₈alkyl-(C₃-C₈ heterocycle);R⁵ is selected from H and methyl;or R⁴ and R⁵ jointly form a carbocyclic ring and have the formula—(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) are independently selectedfrom H, C₁-C₈ alkyl and C₃-C₈ carbocycle and n is selected from 2, 3, 4,5 and 6;R⁶ is selected from H and C₁-C₈ alkyl;R⁷ is selected from H, C₁-C₈ alkyl, C₃-C₈ carbocycle, aryl, C₁-C₈alkyl-aryl, C₁-C₈ alkyl-(C₃-C₈ carbocycle), C₃-C₈ heterocycle and C₁-C₈alkyl-(C₃-C₈ heterocycle);each R⁸ is independently selected from H, OH, C₁-C₈ alkyl, C₃-C₈carbocycle and O—(C₁-C₈ alkyl);R⁹ is selected from H and C₁-C₈ alkyl;R¹⁰ is selected from aryl or C₃-C₈ heterocycle;Z is O, S, NH, or NR¹², wherein R¹² is C₁-C₈ alkyl;R¹¹ is selected from H, C₁-C₂₀ alkyl, aryl, C₃-C₈ heterocycle,—(R¹³O)_(m)—R¹⁴, or —(R¹³O)_(m)—CH(R¹⁵)₂;m is an integer ranging from 1-1000;R¹³ is C₂-C₈ alkyl;R¹⁴ is H or C₁-C₈ alkyl;each occurrence of R¹⁵ is independently H, COOH, —(CH₂)_(n)—N(R¹⁶)₂,—(CH₂)_(n)—SO₃H, or —(CH₂)_(n)—SO₃—C₁-C₈ alkyl;each occurrence of R¹⁶ is independently H, C₁-C₈ alkyl, or—(CH₂)_(n)—COOH;R¹⁸ is selected from —C(R⁸)₂—C(R⁸)₂-aryl, —C(R⁸)₂—C(R⁸)₂—(C₃-C₈heterocycle), and —C(R⁸)₂—C(R⁸)₂—(C₃-C₈ carbocycle); andn is an integer ranging from 0 to 6.

In one embodiment, R³, R⁴ and R⁷ are independently isopropyl orsec-butyl and R⁵ is —H or methyl. In an exemplary embodiment, R³ and R⁴are each isopropyl, R⁵ is —H, and R⁷ is sec-butyl.

In yet another embodiment, R² and R⁶ are each methyl, and R⁹ is —H.

In still another embodiment, each occurrence of R⁸ is —OCH₃.

In some embodiments, R³ and R⁴ are each isopropyl, R² and R⁶ are eachmethyl, R⁵ is —H, R⁷ is sec-butyl, each occurrence of R⁸ is —OCH₃, andR⁹ is —H.

In one embodiment, Z is —O— or —NH—.

In one embodiment, R¹⁰ is aryl.

In an exemplary embodiment, R¹⁰ is -phenyl.

In an exemplary embodiment, when Z is —O—, R¹¹ is —H, methyl or t-butyl.

In one embodiment, when Z is —NH, R¹¹ is —CH(R¹⁵)₂, wherein R¹⁵ is—(CH₂)_(n)—N(R¹⁶)₂, and R¹⁶ is —C₁-C₈ alkyl or —(CH₂)_(n)—COOH.

In another embodiment, when Z is —NH, R¹¹ is —CH(R¹⁵)₂, wherein R¹⁵ is—(CH₂)_(n)—SO₃H.

An exemplary auristatin embodiment of formula D_(E) is MMAE, wherein thewavy line indicates the covalent attachment to a linker (L) of anantibody-drug conjugate:

An exemplary auristatin embodiment of formula D_(F) is MMAF, wherein thewavy line indicates the covalent attachment to a linker (L) of anantibody-drug conjugate:

Other exemplary embodiments include monomethylvaline compounds havingphenylalanine carboxy modifications at the C-terminus of thepentapeptide auristatin drug moiety (WO 2007/008848) andmonomethylvaline compounds having phenylalanine sidechain modificationsat the C-terminus of the pentapeptide auristatin drug moiety (WO2007/008603).

Nonlimiting exemplary embodiments of ADC of Formula I comprising MMAE orMMAF and various linker components have the following structures andabbreviations (wherein “Ab” is an antibody; p is 1 to about 8, “Val-Cit”is a valine-citrulline dipeptide; and “S” is a sulfur atom:

Nonlimiting exemplary embodiments of ADCs of Formula I comprising MMAFand various linker components further include Ab-MC-PAB-MMAF andAb-PAB-MMAF Immunoconjugates comprising MMAF attached to an antibody bya linker that is not proteolytically cleavable have been shown topossess activity comparable to immunoconjugates comprising MMAF attachedto an antibody by a proteolytically cleavable linker (Doronina et al.(2006) Bioconjugate Chem. 17:114-124). In some such embodiments, drugrelease is believed to be effected by antibody degradation in the cell.

Typically, peptide-based drug moieties can be prepared by forming apeptide bond between two or more amino acids and/or peptide fragments.Such peptide bonds can be prepared, for example, according to a liquidphase synthesis method (see, e.g., E. Schröder and K. Lübke, “ThePeptides”, volume 1, pp 76-136, 1965, Academic Press).Auristatin/dolastatin drug moieties may, in some embodiments, beprepared according to the methods of: U.S. Pat. No. 7,498,298; U.S. Pat.No. 5,635,483; U.S. Pat. No. 5,780,588; Pettit et al (1989) J. Am. Chem.Soc. 111:5463-5465; Pettit et al (1998) Anti-Cancer Drug Design13:243-277; Pettit, G. R., et al. Synthesis, 1996, 719-725; Pettit et al(1996) J. Chem. Soc. Perkin Trans. 1 5:859-863; and Doronina (2003) Nat.Biotechnol. 21(7):778-784.

In some embodiments, auristatin/dolastatin drug moieties of formulasD_(E) such as MMAE, and D_(F), such as MMAF, and drug-linkerintermediates and derivatives thereof, such as MC-MMAF, MC-MMAE,MC-vc-PAB-MMAF, and MC-vc-PAB-MMAE, may be prepared using methodsdescribed in U.S. Pat. No. 7,498,298; Doronina et al. (2006)Bioconjugate Chem. 17:114-124; and Doronina et al. (2003) Nat. Biotech.21:778-784 and then conjugated to an antibody of interest.

(3) Calicheamicin

In some embodiments, the immunoconjugate comprises an antibodyconjugated to one or more calicheamicin molecules. The calicheamicinfamily of antibiotics, and analogues thereof, are capable of producingdouble-stranded DNA breaks at sub-picomolar concentrations (Hinman etal., (1993) Cancer Research 53:3336-3342; Lode et al., (1998) CancerResearch 58:2925-2928). Calicheamicin has intracellular sites of actionbut, in certain instances, does not readily cross the plasma membrane.Therefore, cellular uptake of these agents through antibody-mediatedinternalization may, in some embodiments, greatly enhances theircytotoxic effects. Nonlimiting exemplary methods of preparingantibody-drug conjugates with a calicheamicin drug moiety are described,for example, in U.S. Pat. No. 5,712,374; U.S. Pat. No. 5,714,586; U.S.Pat. No. 5,739,116; and U.S. Pat. No. 5,767,285.

(4) Pyrrolobenzodiazepines

In some embodiments, an ADC comprises a pyrrolobenzodiazepine (PBD). Insome embodiments, PDB dimers recognize and bind to specific DNAsequences. The natural product anthramycin, a PBD, was first reported in1965 (Leimgruber, et al., (1965) J. Am. Chem. Soc., 87:5793-5795;Leimgruber, et al., (1965) J. Am. Chem. Soc., 87:5791-5793). Since then,a number of PBDs, both naturally-occurring and analogues, have beenreported (Thurston, et al., (1994) Chem. Rev. 1994, 433-465 includingdimers of the tricyclic PBD scaffold (U.S. Pat. No. 6,884,799; U.S. Pat.No. 7,049,311; U.S. Pat. No. 7,067,511; U.S. Pat. No. 7,265,105; U.S.Pat. No. 7,511,032; U.S. Pat. No. 7,528,126; U.S. Pat. No. 7,557,099).Without intending to be bound by any particular theory, it is believedthat the dimer structure imparts the appropriate three-dimensional shapefor isohelicity with the minor groove of B-form DNA, leading to a snugfit at the binding site (Kohn, In Antibiotics III. Springer-Verlag, NewYork, pp. 3-11 (1975); Hurley and Needham-VanDevanter, (1986) Acc. Chem.Res., 19:230-237). Dimeric PBD compounds bearing C2 aryl substituentshave been shown to be useful as cytotoxic agents (Hartley et al (2010)Cancer Res. 70(17):6849-6858; Antonow (2010) J. Med. Chem.53(7):2927-2941; Howard et al (2009) Bioorganic and Med. Chem. Letters19(22): 6463-6466).

PBD dimers have been conjugated to antibodies and the resulting ADCshown to have anti-cancer properties. Nonlimiting exemplary linkagesites on the PBD dimer include the five-membered pyrrolo ring, thetether between the PBD units, and the N10-C11 imine group (WO2009/016516; US 2009/304710; US 2010/047257; US 2009/036431; US2011/0256157; WO 2011/130598).

Nonlimiting exemplary PBD dimer components of ADCs are of Formula A:

and salts and solvates thereof, wherein:the wavy line indicates the covalent attachment site to the linker;the dotted lines indicate the optional presence of a double bond betweenC1 and C2 or C2 and C3;

R² is independently selected from H, OH, ═O, ═CH₂, CN, R, OR, ═CH—R¹,═C(R^(D))₂, O—SO₂—R, CO₂R and COR, and optionally further selected fromhalo or dihalo, wherein R^(D) is independently selected from R, CO₂R,COR, CHO, CO₂H, and halo;

R⁶ and R⁹ are independently selected from H, R, OH, OR, SH, SR, NH₂,NHR, NRR′, NO₂, Me₃Sn and halo;

R⁷ is independently selected from H, R, OH, OR, SH, SR, NH₂, NHR, NRR′,NO₂, Me₃Sn and halo; Q is independently selected from O, S and NH;

R¹¹ is either H, or R or, where Q is O, SO₃M, where M is a metal cation;

R and R′ are each independently selected from optionally substitutedC₁₋₈ alkyl, C₁₋₁₂ alkyl, C₃₋₈ heterocyclyl, C₃₋₂₀ heterocycle, and C₅₋₂₀aryl groups, and optionally in relation to the group NRR′, R and R′together with the nitrogen atom to which they are attached form anoptionally substituted 4-, 5-, 6- or 7-membered heterocyclic ring;

R¹², R¹⁶, R¹⁹ and R¹⁷ are as defined for R², R⁶, R⁹ and R⁷ respectively;

R″ is a C₃₋₁₂ alkylene group, which chain may be interrupted by one ormore heteroatoms, e.g. O, S, N(H), NMe and/or aromatic rings, e.g.benzene or pyridine, which rings are optionally substituted; and X andX′ are independently selected from O, S and N(H).

In some embodiments, R and R′ are each independently selected fromoptionally substituted C₁₋₁₂ alkyl, C₃₋₂₀ heterocycle, and C₅₋₂₀ arylgroups, and optionally in relation to the group NRR′, R and R′ togetherwith the nitrogen atom to which they are attached form an optionallysubstituted 4-, 5-, 6- or 7-membered heterocyclic ring.

In some embodiments, R⁹ and R¹⁹ are H.

In some embodiments, R⁶ and R¹⁶ are H.

In some embodiments, R⁷ are R¹⁷ are both OR^(7A), where R^(7A) isoptionally substituted C₁₋₄ alkyl. In some embodiments, R^(7A) is Me. Insome embodiments, R^(7A) is Ch₂Ph, where Ph is a phenyl group.

In some embodiments, X is O.

In some embodiments, R¹¹ is H.

In some embodiments, there is a double bond between C2 and C3 in eachmonomer unit.

In some embodiments, R² and R¹² are independently selected from H and R.In some embodiments, R² and R¹² are independently R. In someembodiments, R² and R¹² are independently optionally substituted C₅₋₂₀aryl or C₅₋₇ aryl or C₈₋₁₀ aryl. In some embodiments, R² and R¹² areindependently optionally substituted phenyl, thienyl, napthyl, pyridyl,quinolinyl, or isoquinolinyl. In some embodiments, R² and R¹² areindependently selected from ═O, ═CH₂, ═CH—R¹, and ═C(R^(D))₂. In someembodiments, R² and R¹² each ═CH₂. In some embodiments, R² and R¹² areeach H. In some embodiments, R² and R¹² are each ═O. In someembodiments, R² and R¹² are each ═CF₂. In some embodiments, R² and/orR¹² are independently ═C(R^(D))₂. In some embodiments, R² and/or R¹² areindependently ═CH—R^(D).

In some embodiments, when R² and/or R¹² is ═CH—R^(D), each group mayindependently have either configuration shown below:

In some embodiments, a═CH—R^(D) is in configuration (I).

In some embodiments, R″ is a C₃ alkylene group or a C₅ alkylene group.

In some embodiments, an exemplary PBD dimer component of an ADC has thestructure of Formula A(I):

wherein n is 0 or 1.

In some embodiments, an exemplary PBD dimer component of an ADC has thestructure of Formula A(II):

wherein n is 0 or 1.

In some embodiments, an exemplary PBD dimer component of an ADC has thestructure of Formula A(III):

wherein R^(E) and R^(E″) are each independently selected from H orR^(D), wherein R^(D) is defined as above; and wherein n is 0 or 1.In some embodiments, n is 0. In some embodiments, n is 1. In someembodiments, R^(E) and/or R^(E″) is H. In some embodiments, R^(E) andR^(E″) are H. In some embodiments, R^(E) and/or R^(E″) is R^(D), whereinR^(D) is optionally substituted C₁₋₁₂ alkyl. In some embodiments, R^(E)and/or R^(E″) is R^(D), wherein R^(D) is methyl.

In some embodiments, an exemplary PBD dimer component of an ADC has thestructure of Formula A(IV):

wherein Ar¹ and Ar² are each independently optionally substituted C₅₋₂₀aryl; wherein Ar¹ and Ar² may be the same or different; andwherein n is 0 or 1.

In some embodiments, an exemplary PBD dimer component of an ADC has thestructure of Formula A(V):

wherein Ar¹ and Ar² are each independently optionally substituted C₅₋₂₀aryl; wherein Ar¹ and Ar² may be the same or different; andwherein n is 0 or 1.

In some embodiments, Ar¹ and Ar² are each independently selected fromoptionally substituted phenyl, furanyl, thiophenyl and pyridyl. In someembodiments, Ar¹ and Ar² are each independently optionally substitutedphenyl. In some embodiments, Ar¹ and Ar² are each independentlyoptionally substituted thien-2-yl or thien-3-yl. In some embodiments,Ar¹ and Ar² are each independently optionally substituted quinolinyl orisoquinolinyl. The quinolinyl or isoquinolinyl group may be bound to thePBD core through any available ring position. For example, thequinolinyl may be quinolin-2-yl, quinolin-3-yl, quinolin-4yl,quinolin-5-yl, quinolin-6-yl, quinolin-7-yl and quinolin-8-yl. In someembodiments, the quinolinyl is selected from quinolin-3-yl andquinolin-6-yl. The isoquinolinyl may be isoquinolin-1-yl,isoquinolin-3-yl, isoquinolin-4yl, isoquinolin-5-yl, isoquinolin-6-yl,isoquinolin-7-yl and isoquinolin-8-yl. In some embodiments, theisoquinolinyl is selected from isoquinolin-3-yl and isoquinolin-6-yl.

Further nonlimiting exemplary PBD dimer components of ADCs are ofFormula B:

and salts and solvates thereof, wherein:the wavy line indicates the covalent attachment site to the linker;the wavy line connected to the OH indicates the S or R configuration;R^(V1) and R^(V2) are independently selected from H, methyl, ethyl andphenyl (which phenyl may be optionally substituted with fluoro,particularly in the 4 position) and C₅₋₆ heterocyclyl; wherein R^(V1)and R^(V2) may be the same or different; andn is 0 or 1.

In some embodiments, R^(V1) and R^(V2) are independently selected fromH, phenyl, and 4-fluorophenyl.

In some embodiments, a linker may be attached at one of various sites ofthe PBD dimer drug moiety, including the N10 imine of the B ring, theC-2 endo/exo position of the C ring, or the tether unit linking the Arings (see structures C(I) and C(II) below).

Nonlimiting exemplary PBD dimer components of ADCs include Formulas C(I)and C(II):

Formulas C(I) and C(II) are shown in their N10-C11 imine form. ExemplaryPBD drug moieties also include the carbinolamine and protectedcarbinolamine forms as well, as shown in the table below:

wherein:X is CH₂ (n=1 to 5), N, or O;Z and Z′ are independently selected from OR and NR₂, where R is aprimary, secondary or tertiary alkyl chain containing 1 to 5 carbonatoms;R₁, R′₁, R₂ and R′₂ are each independently selected from H, C₁-C₈ alkyl,C₂-C₈ alkenyl, C₂-C₈ alkynyl, C₅₋₂₀ aryl (including substituted aryls),C₅₋₂₀ heteroaryl groups, —NH₂, —NHMe, —OH, and —SH, where, in someembodiments, alkyl, alkenyl and alkynyl chains comprise up to 5 carbonatoms;R₃ and R′₃ are independently selected from H, OR, NHR, and NR₂, where Ris a primary, secondary or tertiary alkyl chain containing 1 to 5 carbonatoms;R₄ and R′₄ are independently selected from H, Me, and OMe;R₅ is selected from C₁-C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl, C₅₋₂₀aryl (including aryls substituted by halo, nitro, cyano, alkoxy, alkyl,heterocyclyl) and C₅₋₂₀ heteroaryl groups, where, in some embodiments,alkyl, alkenyl and alkynyl chains comprise up to 5 carbon atoms;R₁₁ is H, C₁-C₈ alkyl, or a protecting group (such as acetyl,trifluoroacetyl, t-butoxycarbonyl (BOC), benzyloxycarbonyl (CBZ),9-fluorenylmethylenoxycarbonyl (Fmoc), or a moiety comprising aself-immolating unit such as valine-citrulline-PAB);R₁₂ is H, C₁-C₈ alkyl, or a protecting group;wherein a hydrogen of one of R₁, R′₁, R₂, R′₂, or R₁₂ or a hydrogen ofthe —OCH₂CH₂(X)_(n)CH₂CH₂O— spacer between the A rings is replaced witha bond connected to the linker of the ADC.

Exemplary PDB dimer portions of ADC include, but are not limited to (thewavy line indicates the site of covalent attachment to the linker):

Nonlimiting exemplary embodiments of ADCs comprising PBD dimers have thefollowing structures:

wherein:n is 0 to 12. In some embodiments, n is 2 to 10. In some embodiments, nis 4 to 8. In some embodiments, n is selected from 4, 5, 6, 7, and 8.

The linkers of PBD dimer-val-cit-PAB-Ab and the PBDdimer-Phe-homoLys-PAB-Ab are protease cleavable, while the linker of PBDdimer-maleimide-acetal is acid-labile.

PBD dimers and ADC comprising PBD dimers may be prepared according tomethods known in the art. See, e.g., WO 2009/016516; US 2009/304710; US2010/047257; US 2009/036431; US 2011/0256157; WO 2011/130598.

(5) Anthracyclines

In some embodiments, an ADC comprising anthracycline. Anthracyclines areantibiotic compounds that exhibit cytotoxic activity. While notintending to be bound by any particular theory, studies have indicatedthat anthracyclines may operate to kill cells by a number of differentmechanisms, including: 1) intercalation of the drug molecules into theDNA of the cell thereby inhibiting DNA-dependent nucleic acid synthesis;2) production by the drug of free radicals which then react withcellular macromolecules to cause damage to the cells, and/or 3)interactions of the drug molecules with the cell membrane (see, e.g., C.Peterson et al., “Transport And Storage Of Anthracycline In ExperimentalSystems And Human Leukemia” in Anthracycline Antibiotics In CancerTherapy; N. R. Bachur, “Free Radical Damage” id. at pp. 97-102). Becauseof their cytotoxic potential anthracyclines have been used in thetreatment of numerous cancers such as leukemia, breast carcinoma, lungcarcinoma, ovarian adenocarcinoma and sarcomas (see e.g., P. H-Wiernik,in Anthracycline: Current Status And New Developments p 11).

Nonlimiting exemplary anthracyclines include doxorubicin, epirubicin,idarubicin, daunomycin, nemorubicin, and derivatives thereof.Immunoconjugates and prodrugs of daunorubicin and doxorubicin have beenprepared and studied (Kratz et al (2006) Current Med. Chem. 13:477-523;Jeffrey et al (2006) Bioorganic & Med. Chem. Letters 16:358-362; Torgovet al (2005) Bioconj. Chem. 16:717-721; Nagy et al (2000) Proc. Natl.Acad. Sci. USA 97:829-834; Dubowchik et al (2002) Bioorg. & Med. Chem.Letters 12:1529-1532; King et al (2002) J. Med. Chem. 45:4336-4343; EP0328147; U.S. Pat. No. 6,630,579). The antibody-drug conjugateBR96-doxorubicin reacts specifically with the tumor-associated antigenLewis-Y and has been evaluated in phase I and II studies (Saleh et al(2000) J. Clin. Oncology 18:2282-2292; Ajani et al (2000) Cancer Jour.6:78-81; Tolcher et al (1999) J. Clin. Oncology 17:478-484).

PNU-159682 is a potent metabolite (or derivative) of nemorubicin(Quintieri, et al. (2005) Clinical Cancer Research 11(4):1608-1617).Nemorubicin is a semisynthetic analog of doxorubicin with a2-methoxymorpholino group on the glycoside amino of doxorubicin and hasbeen under clinical evaluation (Grandi et al (1990) Cancer Treat. Rev.17:133; Ripamonti et al (1992) Brit. J. Cancer 65:703;), including phaseII/III trials for hepatocellular carcinoma (Sun et al (2003) Proceedingsof the American Society for Clinical Oncology 22, Abs1448; Quintieri(2003) Proceedings of the American Association of Cancer Research,44:1st Ed, Abs 4649; Pacciarini et al (2006) Jour. Clin. Oncology24:14116).

A nonlimiting exemplary ADC comprising nemorubicin or nemorubicinderivatives is shown in Formula Ia:

wherein R₁ is hydrogen atom, hydroxy or methoxy group and R₂ is a C₁-C₅alkoxy group, or a pharmaceutically acceptable salt thereof;L₁ and Z together are a linker (L) as described herein;T is an antibody (Ab) as described herein; andm is 1 to about 20. In some embodiments, m is 1 to 10, 1 to 7, 1 to 5,or 1 to 4.

In some embodiments, R₁ and R₂ are both methoxy (—OMe).

A further nonlimiting exemplary ADC comprising nemorubicin ornemorubicin derivatives is shown in Formula Ib:

wherein R₁ is hydrogen atom, hydroxy or methoxy group and R₂ is a C₁-C₅alkoxy group, or a pharmaceutically acceptable salt thereof;L₂ and Z together are a linker (L) as described herein;T is an antibody (Ab) as described herein; andm is 1 to about 20. In some embodiments, m is 1 to 10, 1 to 7, 1 to 5,or 1 to 4.

In some embodiments, R₁ and R₂ are both methoxy (—OMe).

In some embodiments, the nemorubicin component of anemorubicin-containing ADC is PNU-159682. In some such embodiments, thedrug portion of the ADC may have one of the following structures:

wherein the wavy line indicates the attachment to the linker (L).

Anthracyclines, including PNU-159682, may be conjugated to antibodiesthrough several linkage sites and a variety of linkers (US 2011/0076287;WO2009/099741; US 2010/0034837; WO 2010/009124), including the linkersdescribed herein.

Exemplary ADCs comprising a nemorubicin and linker include, but are notlimited to:

wherein:R₁ and R₂ are independently selected from H and C₁-C₆ alkyl; and

The linker of PNU-159682 maleimide acetal-Ab is acid-labile, while thelinkers of PNU-159682-val-cit-PAB-Ab, PNU-159682-val-cit-PAB-spacer-Ab,and PNU-159682-val-cit-PAB-spacer(R¹R²)-Ab are protease cleavable.

(6) Other Drug Moieties

Drug moieties also include geldanamycin (Mandler et al (2000) J. Nat.Cancer Inst. 92(19):1573-1581; Mandler et al (2000) Bioorganic & Med.Chem. Letters 10:1025-1028; Mandler et al (2002) Bioconjugate Chem.13:786-791); and enzymatically active toxins and fragments thereof,including, but not limited to, diphtheria A chain, nonbinding activefragments of diphtheria toxin, exotoxin A chain (from Pseudomonasaeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes.See, e.g., WO 93/21232.

Drug moieties also include compounds with nucleolytic activity (e.g., aribonuclease or a DNA endonuclease).

In certain embodiments, an immunoconjugate may comprise a highlyradioactive atom. A variety of radioactive isotopes are available forthe production of radioconjugated antibodies. Examples include At²¹¹,I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, sm¹⁵³, Bi²¹², P³², Pb²¹² and radioactiveisotopes of Lu. In some embodiments, when an immunoconjugate is used fordetection, it may comprise a radioactive atom for scintigraphic studies,for example Tc⁹⁹ or I¹²³, or a spin label for nuclear magnetic resonance(NMR) imaging (also known as magnetic resonance imaging, MRI), such aszirconium-89, iodine-123, iodine-131, indium-111, fluorine-19,carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.Zirconium-89 may be complexed to various metal chelating agents andconjugated to antibodies, e.g., for PET imaging (WO 2011/056983).

The radio- or other labels may be incorporated in the immunoconjugate inknown ways. For example, a peptide may be biosynthesized or chemicallysynthesized using suitable amino acid precursors comprising, forexample, one or more fluorine-19 atoms in place of one or morehydrogens. In some embodiments, labels such as Tc⁹⁹, I¹²³, Re¹⁸⁶, Re¹⁸⁸and In¹¹¹ can be attached via a cysteine residue in the antibody. Insome embodiments, yttrium-90 can be attached via a lysine residue of theantibody. In some embodiments, the IODOGEN method (Fraker et al (1978)Biochem. Biophys. Res. Commun. 80: 49-57 can be used to incorporateiodine-123. “Monoclonal Antibodies in Immunoscintigraphy” (Chatal, CRCPress 1989) describes certain other methods.

In certain embodiments, an immunoconjugate may comprise an antibodyconjugated to a prodrug-activating enzyme. In some such embodiments, aprodrug-activating enzyme converts a prodrug (e.g., a peptidylchemotherapeutic agent, see WO 81/01145) to an active drug, such as ananti-cancer drug. Such immunoconjugates are useful, in some embodiments,in antibody-dependent enzyme-mediated prodrug therapy (“ADEPT”). Enzymesthat may be conjugated to an antibody include, but are not limited to,alkaline phosphatases, which are useful for convertingphosphate-containing prodrugs into free drugs; arylsulfatases, which areuseful for converting sulfate-containing prodrugs into free drugs;cytosine deaminase, which is useful for converting non-toxic5-fluorocytosine into the anti-cancer drug, 5-fluorouracil; proteases,such as serratia protease, thermolysin, subtilisin, carboxypeptidasesand cathepsins (such as cathepsins B and L), which are useful forconverting peptide-containing prodrugs into free drugs;D-alanylcarboxypeptidases, which are useful for converting prodrugs thatcontain D-amino acid substituents; carbohydrate-cleaving enzymes such asβ-galactosidase and neuraminidase, which are useful for convertingglycosylated prodrugs into free drugs; β-lactamase, which is useful forconverting drugs derivatized with β-lactams into free drugs; andpenicillin amidases, such as penicillin V amidase and penicillin Gamidase, which are useful for converting drugs derivatized at theiramine nitrogens with phenoxyacetyl or phenylacetyl groups, respectively,into free drugs. In some embodiments, enzymes may be covalently bound toantibodies by recombinant DNA techniques well known in the art. See,e.g., Neuberger et al., Nature 312:604-608 (1984).

c) Drug Loading

Drug loading is represented by p, the average number of drug moietiesper antibody in a molecule of Formula I. Drug loading may range from 1to 20 drug moieties (D) per antibody. ADCs of Formula I includecollections of antibodies conjugated with a range of drug moieties, from1 to 20. The average number of drug moieties per antibody inpreparations of ADC from conjugation reactions may be characterized byconventional means such as mass spectroscopy, ELISA assay, and HPLC. Thequantitative distribution of ADC in terms of p may also be determined.In some instances, separation, purification, and characterization ofhomogeneous ADC where p is a certain value from ADC with other drugloadings may be achieved by means such as reverse phase HPLC orelectrophoresis.

For some antibody-drug conjugates, p may be limited by the number ofattachment sites on the antibody. For example, where the attachment is acysteine thiol, as in certain exemplary embodiments above, an antibodymay have only one or several cysteine thiol groups, or may have only oneor several sufficiently reactive thiol groups through which a linker maybe attached. In certain embodiments, higher drug loading, e.g. p>5, maycause aggregation, insolubility, toxicity, or loss of cellularpermeability of certain antibody-drug conjugates. In certainembodiments, the average drug loading for an ADC ranges from 1 to about8; from about 2 to about 6; or from about 3 to about 5. Indeed, it hasbeen shown that for certain ADCs, the optimal ratio of drug moieties perantibody may be less than 8, and may be about 2 to about 5 (U.S. Pat.No. 7,498,298).

In certain embodiments, fewer than the theoretical maximum of drugmoieties are conjugated to an antibody during a conjugation reaction. Anantibody may contain, for example, lysine residues that do not reactwith the drug-linker intermediate or linker reagent, as discussed below.Generally, antibodies do not contain many free and reactive cysteinethiol groups which may be linked to a drug moiety; indeed most cysteinethiol residues in antibodies exist as disulfide bridges. In certainembodiments, an antibody may be reduced with a reducing agent such asdithiothreitol (DTT) or tricarbonylethylphosphine (TCEP), under partialor total reducing conditions, to generate reactive cysteine thiolgroups. In certain embodiments, an antibody is subjected to denaturingconditions to reveal reactive nucleophilic groups such as lysine orcysteine.

The loading (drug/antibody ratio) of an ADC may be controlled indifferent ways, and for example, by: (i) limiting the molar excess ofdrug-linker intermediate or linker reagent relative to antibody, (ii)limiting the conjugation reaction time or temperature, and (iii) partialor limiting reductive conditions for cysteine thiol modification.

It is to be understood that where more than one nucleophilic groupreacts with a drug-linker intermediate or linker reagent, then theresulting product is a mixture of ADC compounds with a distribution ofone or more drug moieties attached to an antibody. The average number ofdrugs per antibody may be calculated from the mixture by a dual ELISAantibody assay, which is specific for antibody and specific for thedrug. Individual ADC molecules may be identified in the mixture by massspectroscopy and separated by HPLC, e.g. hydrophobic interactionchromatography (see, e.g., McDonagh et al (2006) Prot. Engr. Design &Selection 19(7):299-307; Hamblett et al (2004) Clin. Cancer Res.10:7063-7070; Hamblett, K. J., et al. “Effect of drug loading on thepharmacology, pharmacokinetics, and toxicity of an anti-CD30antibody-drug conjugate,” Abstract No. 624, American Association forCancer Research, 2004 Annual Meeting, Mar. 27-31, 2004, Proceedings ofthe AACR, Volume 45, March 2004; Alley, S. C., et al. “Controlling thelocation of drug attachment in antibody-drug conjugates,” Abstract No.627, American Association for Cancer Research, 2004 Annual Meeting, Mar.27-31, 2004, Proceedings of the AACR, Volume 45, March 2004). In certainembodiments, a homogeneous ADC with a single loading value may beisolated from the conjugation mixture by electrophoresis orchromatography.

d) Certain Methods of Preparing Immunoconjugates

An ADC of Formula I may be prepared by several routes employing organicchemistry reactions, conditions, and reagents known to those skilled inthe art, including: (1) reaction of a nucleophilic group of an antibodywith a bivalent linker reagent to form Ab-L via a covalent bond,followed by reaction with a drug moiety D; and (2) reaction of anucleophilic group of a drug moiety with a bivalent linker reagent, toform D-L, via a covalent bond, followed by reaction with a nucleophilicgroup of an antibody. Exemplary methods for preparing an ADC of FormulaI via the latter route are described in U.S. Pat. No. 7,498,298, whichis expressly incorporated herein by reference.

Nucleophilic groups on antibodies include, but are not limited to: (i)N-terminal amine groups, (ii) side chain amine groups, e.g. lysine,(iii) side chain thiol groups, e.g. cysteine, and (iv) sugar hydroxyl oramino groups where the antibody is glycosylated Amine, thiol, andhydroxyl groups are nucleophilic and capable of reacting to formcovalent bonds with electrophilic groups on linker moieties and linkerreagents including: (i) active esters such as NHS esters, HOBt esters,haloformates, and acid halides; (ii) alkyl and benzyl halides such ashaloacetamides; and (iii) aldehydes, ketones, carboxyl, and maleimidegroups. Certain antibodies have reducible interchain disulfides, i.e.cysteine bridges. Antibodies may be made reactive for conjugation withlinker reagents by treatment with a reducing agent such as DTT(dithiothreitol) or tricarbonylethylphosphine (TCEP), such that theantibody is fully or partially reduced. Each cysteine bridge will thusform, theoretically, two reactive thiol nucleophiles. Additionalnucleophilic groups can be introduced into antibodies throughmodification of lysine residues, e.g., by reacting lysine residues with2-iminothiolane (Traut's reagent), resulting in conversion of an amineinto a thiol. Reactive thiol groups may also be introduced into anantibody by introducing one, two, three, four, or more cysteine residues(e.g., by preparing variant antibodies comprising one or more non-nativecysteine amino acid residues).

Antibody-drug conjugates provided herein may also be produced byreaction between an electrophilic group on an antibody, such as analdehyde or ketone carbonyl group, with a nucleophilic group on a linkerreagent or drug. Useful nucleophilic groups on a linker reagent include,but are not limited to, hydrazide, oxime, amino, hydrazine,thiosemicarbazone, hydrazine carboxylate, and arylhydrazide. In oneembodiment, an antibody is modified to introduce electrophilic moietiesthat are capable of reacting with nucleophilic substituents on thelinker reagent or drug. In another embodiment, the sugars ofglycosylated antibodies may be oxidized, e.g. with periodate oxidizingreagents, to form aldehyde or ketone groups which may react with theamine group of linker reagents or drug moieties. The resulting imineSchiff base groups may form a stable linkage, or may be reduced, e.g. byborohydride reagents to form stable amine linkages. In one embodiment,reaction of the carbohydrate portion of a glycosylated antibody witheither galactose oxidase or sodium meta-periodate may yield carbonyl(aldehyde and ketone) groups in the antibody that can react withappropriate groups on the drug (Hermanson, Bioconjugate Techniques). Inanother embodiment, antibodies containing N-terminal serine or threonineresidues can react with sodium meta-periodate, resulting in productionof an aldehyde in place of the first amino acid (Geoghegan & Stroh,(1992) Bioconjugate Chem. 3:138-146; U.S. Pat. No. 5,362,852). Such analdehyde can be reacted with a drug moiety or linker nucleophile.

Exemplary nucleophilic groups on a drug moiety include, but are notlimited to: amine, thiol, hydroxyl, hydrazide, oxime, hydrazine,thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groupscapable of reacting to form covalent bonds with electrophilic groups onlinker moieties and linker reagents including: (i) active esters such asNHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl andbenzyl halides such as haloacetamides; (iii) aldehydes, ketones,carboxyl, and maleimide groups.

Nonlimiting exemplary cross-linker reagents that may be used to prepareADC are described herein in the section titled “Exemplary Linkers.”Methods of using such cross-linker reagents to link two moieties,including a proteinaceous moiety and a chemical moiety are known in theart. In some embodiments, a fusion protein comprising an antibody and acytotoxic agent may be made, e.g., by recombinant techniques or peptidesynthesis. A recombinant DNA molecule may comprise regions encoding theantibody and cytotoxic portions of the conjugate either adjacent to oneanother or separated by a region encoding a linker peptide which doesnot destroy the desired properties of the conjugate.

In yet another embodiment, an antibody may be conjugated to a “receptor”(such as streptavidin) for utilization in tumor pre-targeting whereinthe antibody-receptor conjugate is administered to the patient, followedby removal of unbound conjugate from the circulation using a clearingagent and then administration of a “ligand” (e.g., avidin) which isconjugated to a cytotoxic agent (e.g., a drug or radionucleotide).

E. Methods and Compositions for Diagnostics and Detection

In certain embodiments, any of the anti-FcRH5 antibodies provided hereinis useful for detecting the presence of FcRH5 (e.g., FcRH5) in abiological sample. The term “detecting” as used herein encompassesquantitative or qualitative detection. In certain embodiments, abiological sample comprises a cell or tissue. In certain embodiments,such tissues include normal and/or cancerous tissues that express FcRH5at higher levels relative to other tissues, for example, B-cells and/orB-cell associated tissues. In some embodiments, the anti-FcRH5 antibodybinds an isoform c-specific region of the extracellular domain ofFcRH5c. In some embodiments, the anti-FcRH5 antibodies binds Ig-likedomain 9 of FcRH5c.

In one aspect, provided herein are methods of detecting the presence ofFcRH5 in a biological sample. In certain embodiments, the methodcomprises contacting the biological sample with an anti-FcRH5 antibodyunder conditions permissive for binding of the anti-FcRH5 antibody toFcRH5, and detecting whether a complex is formed between the anti-FcRH5antibody and FcRH5. In one aspect, the invention provides a method ofdiagnosing a disorder associated with increased expression of FcRH5. Incertain embodiments, the method comprises contacting a test cell with ananti-FcRH5 antibody; determining the level of expression (eitherquantitatively or qualitatively) of FcRH5 by the test cell by detectingbinding of the anti-FcRH5 antibody to FcRH5; and comparing the level ofexpression of FcRH5 by the test cell with the level of expression ofFcRH5 by a control cell (e.g., a normal cell of the same tissue originas the test cell or a cell that expresses FcRH5 at levels comparable tosuch a normal cell), wherein a higher level of expression of FcRH5 bythe test cell as compared to the control cell indicates the presence ofa disorder associated with increased expression of FcRH5. In certainembodiments, the test cell is obtained from an individual suspected ofhaving a disorder associated with increased expression of FcRH5. Incertain embodiments, the disorder is a cell proliferative disorder, suchas a cancer or a tumor. In some embodiments, the FcRH5 is FcRH5c. Insome embodiments, the anti-FcRH5 antibody binds an isoform c-specificregion of the extracellular domain of FcRH5c. In some embodiments, theanti-FcRH5 antibodies binds Ig-like domain 9 of FcRH5c.

Exemplary cell proliferative disorders that may be diagnosed using anantibody described herein include a B-cell disorder and/or a B-cellproliferative disorder including, but not limited to, lymphoma, multiplemyeloma non-Hodgkin's lymphoma (NHL), aggressive NHL, relapsedaggressive NHL, relapsed indolent NHL, refractory NHL, refractoryindolent NHL, chronic lymphocytic leukemia (CLL), small lymphocyticlymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocyticleukemia (ALL), and mantle cell lymphoma.

In one embodiment, an anti-FcRH5 antibody for use in a method ofdiagnosis or detection is provided. In a further aspect, a method ofdetecting the presence of FcRH5 in a biological sample is provided. Incertain embodiments, the method comprises contacting the biologicalsample with an anti-FcRH5 antibody as described herein under conditionspermissive for binding of the anti-FcRH5 antibody to FcRH5, anddetecting whether a complex is formed between the anti-FcRH5 antibodyand FcRH5 in the biological sample. Such method may be an in vitro or invivo method. In one embodiment, an anti-FcRH5 antibody is used to selectsubjects eligible for therapy with an anti-FcRH5 antibody, e.g. whereFcRH5 is a biomarker for selection of patients. In a further embodiment,the biological sample is a cell or tissue (e.g., biopsy material). Insome embodiments, the anti-FcRH5 antibody binds an isoform c-specificregion of the extracellular domain of FcRH5c. In some embodiments, theanti-FcRH5 antibodies binds Ig-like domain 9 of FcRH5c.

In a further embodiment, an anti-FcRH5 antibody is used in vivo todetect, e.g., by in vivo imaging, an FcRH5-positive cancer in a subject,e.g., for the purposes of diagnosing, prognosing, or staging cancer,determining the appropriate course of therapy, or monitoring response ofa cancer to therapy. One method known in the art for in vivo detectionis immuno-positron emission tomography (immuno-PET), as described, e.g.,in van Dongen et al., The Oncologist 12:1379-1389 (2007) and Verel etal., J. Nucl. Med. 44:1271-1281 (2003). In such embodiments, a method isprovided for detecting an FcRH5-positive cancer in a subject, the methodcomprising administering a labeled anti-FcRH5 antibody to a subjecthaving or suspected of having an FcRH5-positive cancer, and detectingthe labeled anti-FcRH5 antibody in the subject, wherein detection of thelabeled anti-FcRH5 antibody indicates an FcRH5-positive cancer in thesubject. In certain of such embodiments, the labeled anti-FcRH5 antibodycomprises an anti-FcRH5 antibody conjugated to a positron emitter, suchas ⁶⁸Ga, ¹⁸F, ⁶⁴Cu, ⁸⁶Y, ⁷⁶Br, ⁸⁹Zr, and ¹²⁴I. In a particularembodiment, the positron emitter is ⁸⁹Zr. In some embodiments, theanti-FcRH5 antibody binds an isoform c-specific region of theextracellular domain of FcRH5c. In some embodiments, the anti-FcRH5antibodies binds Ig-like domain 9 of FcRH5c.

In further embodiments, a method of diagnosis or detection comprisescontacting a first anti-FcRH5 antibody immobilized to a substrate with abiological sample to be tested for the presence of FcRH5, exposing thesubstrate to a second anti-FcRH5 antibody, and detecting whether thesecond anti-FcRH5 is bound to a complex between the first anti-FcRH5antibody and FcRH5 in the biological sample. A substrate may be anysupportive medium, e.g., glass, metal, ceramic, polymeric beads, slides,chips, and other substrates. In certain embodiments, a biological samplecomprises a cell, blood, or tissue (e.g., biopsy material)

Exemplary disorders that may be diagnosed or detected according to anyof the above embodiments include FcRH5-positive cancers, such asFcRH5-positive B-cell proliferative disease, FcRH5-positive plasma cellneoplasm, and FcRH5-positive multiple myeloma. In some embodiments, anFcRH5-positive cancer is detected by anti-FcRH5 immunohistochemistry(IHC) or in situ hybridization (ISH). In some embodiments, anFcRH5-positive cancer is a cancer that expresses FcRH5 according to areverse-transcriptase PCR (RT-PCR) assay that detects FcRH5 mRNA. Insome embodiments, the RT-PCR is quantitative RT-PCR.

In certain embodiments, labeled anti-FcRH5 antibodies are provided. Insome embodiments, the anti-FcRH5 antibody binds an isoform c-specificregion of the extracellular domain of FcRH5c. In some embodiments, theanti-FcRH5 antibodies binds Ig-like domain 9 of FcRH5c. Labels include,but are not limited to, labels or moieties that are detected directly(such as fluorescent, chromophoric, electron-dense, chemiluminescent,and radioactive labels), as well as moieties, such as enzymes orligands, that are detected indirectly, e.g., through an enzymaticreaction or molecular interaction. Exemplary labels include, but are notlimited to, the radioisotopes ³²P, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I, fluorophoressuch as rare earth chelates or fluorescein and its derivatives,rhodamine and its derivatives, dansyl, umbelliferone, luciferases, e.g.,firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456),luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP),alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme,saccharide oxidases, e.g., glucose oxidase, galactose oxidase, andglucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricaseand xanthine oxidase, coupled with an enzyme that employs hydrogenperoxide to oxidize a dye precursor such as HRP, lactoperoxidase, ormicroperoxidase, biotin/avidin, spin labels, bacteriophage labels,stable free radicals, and the like. In another embodiment, a label is apositron emitter. Positron emitters include but are not limited to ⁶⁸Ga,¹⁸F, ⁶⁴Cu, ⁸⁶Br, ⁸⁹Zr, and ¹²⁴I. In a particular embodiment, a positronemitter is ⁸⁹Zr.

F. Pharmaceutical Formulations

Pharmaceutical formulations of an anti-FcRH5 antibody or immunoconjugateas described herein are prepared by mixing such antibody orimmunoconjugate having the desired degree of purity with one or moreoptional pharmaceutically acceptable carriers (Remington'sPharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the formof lyophilized formulations or aqueous solutions. Pharmaceuticallyacceptable carriers are generally nontoxic to recipients at the dosagesand concentrations employed, and include, but are not limited to:buffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride; benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as polyethylene glycol (PEG). Exemplarypharmaceutically acceptable carriers herein further includeinsterstitial drug dispersion agents such as soluble neutral-activehyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, BaxterInternational, Inc.). Certain exemplary sHASEGPs and methods of use,including rHuPH20, are described in US Patent Publication Nos.2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is combined withone or more additional glycosaminoglycanases such as chondroitinases. Insome embodiments, the anti-FcRH5 antibody binds an isoform c-specificregion of the extracellular domain of FcRH5c. In some embodiments, theanti-FcRH5 antibodies binds Ig-like domain 9 of FcRH5c.

Exemplary lyophilized antibody or immunoconjugate formulations aredescribed in U.S. Pat. No. 6,267,958. Aqueous antibody orimmunoconjugate formulations include those described in U.S. Pat. No.6,171,586 and WO2006/044908, the latter formulations including ahistidine-acetate buffer.

The formulation herein may also contain more than one active ingredientas necessary for the particular indication being treated, preferablythose with complementary activities that do not adversely affect eachother.

Active ingredients may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980).

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody and/or immunoconjugate,which matrices are in the form of shaped articles, e.g. films, ormicrocapsules.

The formulations to be used for in vivo administration are generallysterile. Sterility may be readily accomplished, e.g., by filtrationthrough sterile filtration membranes.

G. Therapeutic Methods and Compositions

Any of the anti-FcRH5 antibodies (e.g., FcRH5 bispecific antibodies)and/or immunoconjugates provided herein may be used in methods, e.g.,therapeutic methods. In some embodiments, the anti-FcRH5 antibody bindsan isoform c-specific region of the extracellular domain of FcRH5c. Insome embodiments, the anti-FcRH5 antibodies binds Ig-like domain 9 ofFcRH5c.

In one aspect, an anti-FcRH5 antibody (e.g., FcRH5 bispecific antibody)and/or immunoconjugate provided herein is used in a method of inhibitingproliferation of an FcRH5-positive cell, the method comprising exposingthe cell to the anti-FcRH5 antibody (e.g., FcRH5 bispecific antibody)and/or immunoconjugate under conditions permissive for binding of theanti-FcRH5 antibody (e.g., FcRH5 bispecific antibody) and/orimmunoconjugate to FcRH5 (e.g., FcRH5c) on the surface of the cell,thereby inhibiting the proliferation of the cell. In certainembodiments, the method is an in vitro or an in vivo method. In furtherembodiments, the cell is a B-cell proliferative disorder. In certainembodiments, the cell proliferative disorder is associated withincreased expression and/or activity of FcRH5 (e.g., FcRH5c). Forexample, in certain embodiments, the B-cell proliferative disorder isassociated with increased expression of FcRH5 on the surface of aB-cell. In certain embodiments, the B-cell proliferative disorder is atumor or a cancer. In some embodiments, the B-cell proliferativedisorder is a plasma cell neoplasm. In some embodiments, the plasma cellneoplasm is multiple myeloma, plasmacytoma, and/or MGUS. Examples ofB-cell proliferative disorders to be treated by the antibodies and/orimmunoconjugates of the invention include, but are not limited to,lymphoma, multiple myelomanon-Hodgkins lymphoma (NHL), aggressive NHL,relapsed aggressive NHL, relapsed indolent NHL, refractory NHL,refractory indolent NHL, chronic lymphocytic leukemia (CLL), smalllymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acutelymphocytic leukemia (ALL), and/or mantle cell lymphoma.

Presence of various biomarkers in a sample can be analyzed by a numberof methodologies, many of which are known in the art and understood bythe skilled artisan, including, but not limited to, immunohistochemistry(“IHC”), Western blot analysis, immunoprecipitation, molecular bindingassays, ELISA, ELIFA, fluorescence activated cell sorting (“FACS”),MassARRAY, proteomics, quantitative blood based assays (as for exampleSerum ELISA), biochemical enzymatic activity assays, in situhybridization,

Southern analysis, Northern analysis, whole genome sequencing,polymerase chain reaction (“PCR”) including quantitative real time PCR(“qRT-PCR”) and other amplification type detection methods, such as, forexample, branched DNA, SISBA, TMA and the like, RNA-Seq, FISH,microarray analysis, gene expression profiling, and/or serial analysisof gene expression (“SAGE”), as well as any one of the wide variety ofassays that can be performed by protein, gene, and/or tissue arrayanalysis. Typical protocols for evaluating the status of genes and geneproducts are found, for example in Ausubel et al., eds., 1995, CurrentProtocols In Molecular Biology, Units 2 (Northern Blotting), 4 (SouthernBlotting), 15 (Immunoblotting) and 18 (PCR Analysis). Multiplexedimmunoassays such as those available from Rules Based Medicine or MesoScale Discovery (“MSD”) may also be used.

Inhibition of cell proliferation in vitro may be assayed using theCellTiter-Glo™ Luminescent Cell Viability Assay, which is commerciallyavailable from Promega (Madison, Wis.). That assay determines the numberof viable cells in culture based on quantitation of ATP present, whichis an indication of metabolically active cells. See Crouch et al. (1993)J. Immunol. Meth. 160:81-88, U.S. Pat. No. 6,602,677. The assay may beconducted in 96- or 384-well format, making it amenable to automatedhigh-throughput screening (HTS). See Cree et al. (1995) AntiCancer Drugs6:398-404. The assay procedure involves adding a single reagent(CellTiter-Glo® Reagent) directly to cultured cells. This results incell lysis and generation of a luminescent signal produced by aluciferase reaction. The luminescent signal is proportional to theamount of ATP present, which is directly proportional to the number ofviable cells present in culture. Data can be recorded by luminometer orCCD camera imaging device. The luminescence output is expressed asrelative light units (RLU).

In another aspect, an anti-FcRH5 antibody (e.g., FcRH5 bispecificantibody) and/or immunoconjugate for use as a medicament is provided. Infurther aspects, an anti-FcRH5 antibody (e.g., FcRH5 bispecificantibody) and/or immunoconjugate for use in a method of treatment isprovided. In certain embodiments, an anti-FcRH5 antibody (e.g., FcRH5bispecific antibody) and/or immunoconjugate for use in treating FcRH5(e.g., FcRH5c)-positive cancer is provided. In certain embodiments,provided herein the anti-FcRH5 antibody (including FcRH5 bispecificantibody) and/or immunoconjugate for use in a method of treating anindividual having an FcRH5 (e.g., FcRH5c)-positive cancer, the methodcomprising administering to the individual an effective amount of theanti-FcRH5 antibody and/or immunoconjugate. In some embodiments, theanti-FcRH5 antibody binds an isoform c-specific region of theextracellular domain of FcRH5c. In some embodiments, the anti-FcRH5antibodies binds Ig-like domain 9 of FcRH5c. In one such embodiment, themethod further comprises administering to the individual an effectiveamount of at least one additional therapeutic agent, e.g., as describedbelow.

In a further aspect, provided herein are uses of an anti-FcRH5 antibody(e.g., FcRH5 bispecific antibody) and/or immunoconjugate in themanufacture or preparation of a medicament. In one embodiment, themedicament is for treatment of FcRH5 (e.g., FcRH5c)-positive cancer. Ina further embodiment, the medicament is for use in a method of treatingFcRH5 (e.g., FcRH5c)-positive cancer, the method comprisingadministering to an individual having FcRH5 (e.g., FcRH5c)-positivecancer an effective amount of the medicament. In one such embodiment,the method further comprises administering to the individual aneffective amount of at least one additional therapeutic agent, e.g., asdescribed below. In some embodiments, the anti-FcRH5 antibody binds anisoform c-specific region of the extracellular domain of FcRH5c. In someembodiments, the anti-FcRH5 antibodies binds Ig-like domain 9 of FcRH5c.

In a further aspect, provided herein are methods for treating FcRH5(e.g., FcRH5c)-positive cancer. In one embodiment, the method comprisesadministering to an individual having such FcRH5 (e.g., FcRH5c)-positivecancer an effective amount of an anti-FcRH5 antibody (e.g., FcRH5bispecific antibody) and/or immunoconjugate. In one such embodiment, themethod further comprises administering to the individual an effectiveamount of at least one additional therapeutic agent, as described below.In some embodiments, the anti-FcRH5 antibody binds an isoform c-specificregion of the extracellular domain of FcRH5c. In some embodiments, theanti-FcRH5 antibodies binds Ig-like domain 9 of FcRH5c.

An FcRH5-positive cancer according to any of the above embodiments maybe, e.g., FcRH5-positive B-cell proliferative disorder, FcRH5-positiveplasma cell neoplasm, and/or FcRH5-positive multiple myeloma. In someembodiments, an FcRH5-positive cancer is detected by anti-FcRH5immunohistochemistry (IHC) or in situ hybridization (ISH). In someembodiments, an FcRH5-positive cancer is a cancer that expresses FcRH5according to a reverse-transcriptase PCR (RT-PCR) assay that detectsFcRH5 mRNA. In some embodiments, the RT-PCR is quantitative RT-PCR.

In some embodiments of any of the above embodiments, the individual maybe a human.

In a further aspect, provided herein are pharmaceutical formulationscomprising any of the anti-FcRH5 antibodies and/or immunoconjugateprovided herein, e.g., for use in any of the above therapeutic methods.In one embodiment, a pharmaceutical formulation comprises any of theanti-FcRH5 antibodies (e.g., bispecific antibodies) and/orimmunoconjugates provided herein and a pharmaceutically acceptablecarrier. In another embodiment, a pharmaceutical formulation comprisesany of the anti-FcRH5 antibodies (e.g., bispecific antibodies) and/orimmunoconjugates provided herein and at least one additional therapeuticagent, e.g., as described below.

Antibodies (e.g., bispecific antibodies) and/or immunoconjugatesprovided herein can be used either alone or in combination with otheragents in a therapy. Such combination therapies noted above encompasscombined administration (where two or more therapeutic agents areincluded in the same or separate formulations), and separateadministration, in which case, administration of the antibody orimmunoconjugate provided herein can occur prior to, simultaneously,and/or following, administration of the additional therapeutic agentand/or adjuvant. Antibodies and/or immunoconjugates provided herein canalso be used in combination with radiation therapy.

An antibody (including bispecific antibody) and/or immunoconjugateprovided herein (and any additional therapeutic agent) can beadministered by any suitable means, including parenteral,intrapulmonary, and intranasal, and, if desired for local treatment,intralesional administration. Parenteral infusions includeintramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. Dosing can be by any suitable route, e.g.by injections, such as intravenous or subcutaneous injections, dependingin part on whether the administration is brief or chronic. Variousdosing schedules including but not limited to single or multipleadministrations over various time-points, bolus administration, andpulse infusion are contemplated herein.

Antibodies (e.g., bispecific antibodies) and/or immunoconjugatesprovided herein would be formulated, dosed, and administered in afashion consistent with good medical practice. Factors for considerationin this context include the particular disorder being treated, theparticular mammal being treated, the clinical condition of theindividual patient, the cause of the disorder, the site of delivery ofthe agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. Theantibody (e.g., bispecific antibodies) and/or immunoconjugate need notbe, but is optionally formulated with one or more agents currently usedto prevent or treat the disorder in question. The effective amount ofsuch other agents depends on the amount of antibody or immunoconjugatepresent in the formulation, the type of disorder or treatment, and otherfactors discussed above. These are generally used in the same dosagesand with administration routes as described herein, or about from 1 to99% of the dosages described herein, or in any dosage and by any routethat is empirically/clinically determined to be appropriate.

For the prevention or treatment of disease, the appropriate dosage of anantibody (e.g., bispecific antibody) and/or immunoconjugate providedherein (when used alone or in combination with one or more otheradditional therapeutic agents) will depend on the type of disease to betreated, the type of antibody or immunoconjugate, the severity andcourse of the disease, whether the antibody (e.g., bispecific antibody)and/or immunoconjugate is administered for preventive or therapeuticpurposes, previous therapy, the patient's clinical history and responseto the antibody or immunoconjugate, and the discretion of the attendingphysician. The antibody (e.g., bispecific antibody) and/orimmunoconjugate are suitably administered to the patient at one time orover a series of treatments. Depending on the type and severity of thedisease, about 1 μg/kg to 15 mg/kg (e.g. 0.1 mg/kg-10 mg/kg) of antibodyor immunoconjugate can be an initial candidate dosage for administrationto the patient, whether, for example, by one or more separateadministrations, or by continuous infusion. One typical daily dosagemight range from about 1 μg/kg to 100 mg/kg or more, depending on thefactors mentioned above. For repeated administrations over several daysor longer, depending on the condition, the treatment would generally besustained until a desired suppression of disease symptoms occurs. Oneexemplary dosage of the antibody (e.g., bispecific antibody) and/orimmunoconjugate would be in the range from about 0.05 mg/kg to about 10mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kgor 10 mg/kg (or any combination thereof) may be administered to thepatient. Such doses may be administered intermittently, e.g. every weekor every three weeks (e.g. such that the patient receives from about twoto about twenty, or e.g. about six doses of the antibody). An initialhigher loading dose, followed by one or more lower doses may beadministered. However, other dosage regimens may be useful. The progressof this therapy is easily monitored by conventional techniques andassays.

It is understood that any of the above formulations or therapeuticmethods may be carried out using both an immunoconjugate provided hereinand an anti-FcRH5 antibody. In some embodiments, the anti-FcRH5 antibodybinds an isoform c-specific region of the extracellular domain ofFcRH5c. In some embodiments, the anti-FcRH5 antibodies binds Ig-likedomain 9 of FcRH5c.

H. Articles of Manufacture

In another aspect provided herein, an article of manufacture containingmaterials useful for the treatment, prevention and/or diagnosis of thedisorders described above is provided. The article of manufacturecomprises a container and a label or package insert on or associatedwith the container. Suitable containers include, for example, bottles,vials, syringes, IV solution bags, etc. The containers may be formedfrom a variety of materials such as glass or plastic. The containerholds a composition which is by itself or combined with anothercomposition effective for treating, preventing and/or diagnosing thedisorder and may have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). At least one active agent in thecomposition is an antibody or immunoconjugate provided herein. The labelor package insert indicates that the composition is used for treatingthe condition of choice. Moreover, the article of manufacture maycomprise (a) a first container with a composition contained therein,wherein the composition comprises an FcRH5 antibody (e.g., bispecificantibody) and/or FcRH5 immunoconjugate provided herein; and (b) a secondcontainer with a composition contained therein, wherein the compositioncomprises a further cytotoxic or otherwise therapeutic agent. Thearticle of manufacture in this embodiment provided herein may furthercomprise a package insert indicating that the compositions can be usedto treat a particular condition. Alternatively, or additionally, thearticle of manufacture may further comprise a second (or third)container comprising a pharmaceutically-acceptable buffer, such asbacteriostatic water for injection (BWFI), phosphate-buffered saline,Ringer's solution or dextrose solution. It may further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, and syringes. In someembodiments, the anti-FcRH5 antibody binds an isoform c-specific regionof the extracellular domain of FcRH5c. In some embodiments, theanti-FcRH5 antibodies binds Ig-like domain 9 of FcRH5c.

III. Examples

The following are examples of methods and compositions of the invention.It is understood that various other embodiments may be practiced, giventhe general description provided above.

Materials and Methods

Immunogen (E11-Flag)

Amino acids 745-850 of human FcRH5c (SEQ ID NO:1) were cloned intomammalian expression vector pRK5.NT.Flag using standard protocols andexpressed transiently in CHO cells. The recombinant protein withN-terminal Flag-expression tag was purified using anti-flag and sizeexclusion chromatography on an 5200 Superdex column

Development and Characterization of Mouse Anti-FcRH5 E11 Antibodies

Balb/c mice (Charles River, Hollister, Calif.) were immunized with 2 μghuman FcRH5 E11 ECD protein (amino acid residues 743-850 of SEQ ID NO:1)(Genentech, South San Francisco, Calif.) mixed with MPL+TDM (Ribi)adjuvant via footpad injection. Mice received nine doses, followed by aprefusion boost in PBS alone via footpad and IV routes three days priorto fusion.

Popliteal lymph nodes were harvested and lymphocytes from these mice,all of whose sera demonstrated strong binding titers to the immunizationprotein by ELISA and showed strong FACS reactivity to SVT2 cellstransfected with the human FcRH5 E11 ECD, were fused with X63-Ag8.653mouse myeloma cells (American Type Culture Collection, Rockville, Md.)via electrofusion (Harvard Apparatus, Holliston, Mass.). Fused cellswere incubated at 37° C., 7% CO₂, overnight in Medium C (StemCellTechnologies, Vancouver, BC, Canada), before resuspension in semi-solidMedium D (StemCell Technologies) containing 0.01 mg/ml FITC labeledanti-mouse IgG (Jackson ImmunoResearch, West Grove, Pa.) and platinginto Omniwell trays (Thermo Fisher Scientific, Rochester, N.Y.). Ninedays after plating, fluorescent colonies were selected and transferredinto 96-well plates containing Medium E (StemCell Technologies) using aClonepix FL (Genetix, New Milton, Hampshire, UK). Supernatants werescreened by ELISA against anti-mouse IgG (MP Biomedicals, Santa Ana,Calif.) seven days after picking.

Hybridomas demonstrating mouse IgG expression by ELISA were expanded andscreened by FACS against SVT2 cells overexpressing full-length humanFcRH5, cyno FcRH5, and human FcRH5 E11 ECD. Strong FACS positive cloneswere subcloned by single-cell sorting using a FACSAria (BD, FranklinLakes, N.J.). Final clones demonstrating the highest ELISA and FACSbinding of interest after one or two rounds of subcloning were expandedfor large-scale production in bioreactors (Integra Biosciences, Chur,Switzerland). Supernatants were then purified by Protein A affinitychromatography as previously described (Hongo et al. 2000).

Production of BisFabs

BisFabs were generated by crosslinking a Fab′ of the anti-FcRH5 Mab to aFab′ of the anti-CD3 (UCHT1.v9) Mab at the hinge cysteine residues. Togenerate the Fab′ 2 fragments from the hybridoma Abs different digestionconditions were used: Abs of the mIgG1 isotype were digested with 1:50(w/w) pepsin at pH 3.5 for 1-2 H at 37° C.; mouse IgG2a Abs weredigested with Lysin C endopeptidase at 1 1:500 (w/w) ratio, pH 8, for2-4 h at 37° C.; and mouse IgG2b Abs were digested with Lysin C at a1:100 (w/w) ratio overnight at 37° C. In all cases the F(ab′)₂ fragmentwas isolated from the reaction mixture by capture with a SP column andelution with 10 column volumes of a lineal gradient (0-100%) of 1Msodium chloride. Under the digestion conditions mentioned above mIgG1and mIgG2b produced a F(ab′)₂ fragment containing three Cysteineresidues in the hinge, while the F(ab′)2 from mIgG2a showed two cysteineresidues in the hinge. To generate Fab′ with a single reactive Cys twodifferent methods were used. For fragments containing an odd (3) numberof hinge cysteines (mIgG1 and mIgG2b) the isolated F(ab′)2s were reducedin 25 mM sodium acetate, pH 5, 150 mM sodium chloride, 2 mM EDTA, 2 mMTCEP for 2-6-H at RT. After the reduction step was complete, the samplewas diluted to 0.2 mg/ml, the pH was raised to 7.5 by adding Tris pH 8and 5 mM dehydroascorbic acid (DHAA) was added to drive re-oxidation ofthe cysteines. After an overnight incubation at room temperature thepresence of reduced Thiols was evaluated by probing with an excess ofNEM and analyzing the MW shift by mass spectrometry. After confirmingthe presence of only one reactive Cysteine per molecule, the Fab′ waspurified by gel filtration to remove small amounts of homodimers.

For F(ab′)2 fragments derived from mIgG2a and containing 2 Cysteineresidues in the hinge, a single reactive Cysteine was produced bypartial blocking with N-ethyl maleimide (NEM) as described in Scheer etal (in press). Briefly, the antibody was digested with pepsin (1% w/w)by treatment in sodium acetate buffer at pH 4.5. After digestion for 1hour, the F(ab′)₂ was isolated from the digestion mixture by capture onan SP-HP cation exchange resin and purified by a 10 CV salt gradient of0-1 M NaCl. The F(ab′)₂ was then reduced with 1 mM TCEP in a buffercontaining 25 mm MES, pH 5.8, 2 mM EDTA, and 300 mM NaCl and the Fabswere oxidized by the addition of 5 mM dehydroacorbic acid (DHAA) toreform the disulfide bond between the heavy chain and light chain.

The effector arm of the bisfabs (UCHT1.v9) was generated by pepsindigestion, partial NEM blocking and conjugation to bismaleimide asdescribed before (Scheer et al; in press). Briefly, the two thiols (cysresidues) at the hinge were then reacted with 1 equivalent ofN-ethylmaleimide (NEM) (Sigma Aldrich). The different anti-FCRH5 Fab'scontaining a single reactive Cysteine were incubated with the anti-CD3Fab′ conjugated to the bismaleimide crosslinker overnight at roomtemperature. The ˜100 kDa crosslinked Fabs were separated from theunreacted species by gel filtration and then characterized by SDS-PAGE,mass spectrometry and analytical size exclusion chromatography.

TDB Expression and Purification

TDBs were produced by two different approaches: co-culture of bacteriaexpressing each of the two antibody arms or by expressing each armseparately and then anneling them in vitro. The strategies have beendescribed in Christoph Spiess et al. 2012 and described inPCT/US10/58958 filed on 31 May 2011, which is incorporated by reference.Briefly for the coculture strategy E. coli expressing anti-CD3 (hole)and Ecoli expressing anti-tumor target (knob) were grown together inshaker flasks at a predetermined ratio such that it produced similaramounts o feach hemimer. The cocultured bacterial broth was thenharvested, the cells disrupted in a microfluidizer and the antibodiespurified by Protein A affinity. It has been observed that duringmicrofluidizing and protein A capture the two arms annealed and formedthe hing inter-chain disulfide bridges (Christoph Spiess et al. 2012).Alternatively, the antibody hemimers were grown separately by high-celldensity fermentation and independently isolated by Protein Achromatography. The purified hemimers were than combined at a 1:1 molarration and inclubated in 50 mM Tris, pH 8.5 in the presence of 2 mM DTTfor 4 hours to allow annealing and the reduction of disulfides in thehing region. Dialysis against the same buffer without DTT for 24-48hours resulted in the formation of the inter-chain disulfide bonds. Forboth production strategies the bispecific antibody was purified fromcontaminants by hydrophobic interaction chromatography (HIC) asdescribed in Christoph Spiess et al. 2012. The resulting material wasanalyzed for endotoxin levels using an Endosafe protable etest systemand when needed, the endotoxin content was reduced by washing theprotein with 0.1% Triton X-114.

TDB Characterization

The molecular weight of the bispecific antibody was analyzed by massspectrometry (LC-ESI/TCF) as described before (Jackman et al. 2010). Theantibodies were also analyzed by analytical size exclusionchromatography in Zenix SEC-300 column (Sepax Technologies USA) using anAgilent 1:100 HPLC system. The presence of residual antibody fragmentswas quantified by electrophoresis using a 2100 Bioanalyzer and a Protein230 Chip.

Blood Cell Fractionation

PBMCs were separated from the blood of healthy volunteers usinglymphocyte separation medium (MP biomedicals, Solon, Ohio). CD8+ cellswere extracted from PBMC using human CD8+ Isolation Kit from Miltenyi(#130-094-156) by negative selection.

In Vitro Cytotoxicity Assays (T Cell Killing)

For in vitro cytotoxicity assays 1×10⁴ target cells were plated on 96well plates and incubated overnight. 3×10⁴ CD8+ T-cells were added withor without TDB or BisFab and incubated 48 hours in +37° C. T cells wereremoved by washing twice with growth media. Cell viability was measuredusing CellTiter-Glo® Luminescent Cell Viability Assay (Promega, Madison,Wis.).

Alternatively, in vitro cytotoxicity was monitored by flow cytometry.Target cells were labeled with carboxyfluorescein succinimidyl ester(CFSE) according to manufacturer's protocol (Invitrogen, #C34554). TheCFSE-labeled target cells and purified CD8+ T cells from human PBMC weremixed in 3:1 E:T ratio and incubated with TDB or BisFab for 48 hours. Atthe end of the incubation, the cells were lifted by trypsin andcollected from the plate. The cells were resuspended in equal volume ofPBS+2% FBS+1 mM EDTA+propidium iodine (PI). Flow cytometry analysis wasdone on a FACSCalibur in automation format. The number of live targetcells was counted by gating on CFSE+/PI negative cells. The percentageof cytotoxicity was calculated as follows: % cytotoxicity (live targetcell number w/o TDB−live target cell number w/TDB)/(live target cellnumber w/o TDB)×100.

Analysis of T Cell Activation

Target cells and purified CD8+ T cells were mixed in the presence orabsence of TDB and T cell activation was analyzed by flow cytometry. Atthe end of the incubation, cells were stained with CD8-FITC (BDBiosciences, 555634) and CD69-PE (BD Biosciences, 555531).

Binding of Subclone Supernatants, Monoclonal Antibodies, BisFabs andTDBs

To test binding to endogenously FcRH5 expressing cancer cells or FcRH5transfected cancer cells, cells were lifted using EDTA/PBS. 1×10⁵ cellswere suspended in 100 ul and incubated h with primary antibodies (1volume of non-IgG quantitated subclone supernatant, 4 ug/ml IgGquantified subclone supernatant or 2 ug/ul purified monoclonalantibodies). Cells were washed twice with FACS buffer (PBS 1% BSA 2 mMEDTA) and incubated with 1:1000 dilution of goat anti-mouse secondarylabeled with PE or 1:100 of goat anti-mouse APC. Cells were washed twicewith FACS buffer and Flow cytometry analysis was done on a FACSCalibur.Direct Xenon-labeling of antibodies was done according to manufacturer'sprotocol (Invitrogen), when indicated. To analyze binding to NK or Bcells, 1 million human PBMC were incubated with 4 ug/ml IgG quantifiedsubclone supernatants for 60 min, washed and incubated with 1:100dilution of goat anti-mouse secondary labeled with APC. Cells were thenwashed again twice and stained using anti-CD56 (PE; BD Biosciences#555516) and anti-CD19 (PE; BD Biosciences #340364) prior flow cytometryand analysis of binding to human CD56+ and CD19+ cells.

Results

Initially to produce isoform specific antibodies for the membraneproximal Ig-domain, mice were immunized with recombinant baculovirusproduced E11 protein (amino acids 745-848 of SEQ ID NO:1) of humanFcRH5c and C-terminal His-expression tag). This immunization strategydid not result to significant immune response to FcRH5 and failed toproduce monoclonal anti-FcRH5 antibodies. The second immunizationstrategy was DNA-immunization with plasmid encoding amino acids 745-977of FcRH5c (SEQ ID NO:1) encoding membrane proximal Ig-domain,transmembrane domain and intracellular domains of human FcRH5. Thisimmunization strategy did not result to significant immune response toFcRH5 and failed to produce monoclonal anti-FcRH5 antibodies. The thirdimmunization strategy utilized peptides corresponding to membraneproximal Ig-domain of FcRH5, that were homologous to cyno FcRH5 andnon-homologous to human FcRH1, FcRH2, FcRH3, and FcRH4. Thisimmunization strategy did not result to significant immune response toFcRH5 and failed to produce monoclonal anti-FcRH5 antibodies.

For the fourth immunization strategy E11 protein was produced inCHO-cells consisting of the membrane proximal Ig-domain of human FcRH5(amino acid residues 745-850 of SEQ ID NO:1) with N-terminal Flagexpression tag. The above recombinant protein was used to immunize mice.Immunization, development and characterization of mouse anti-FcRH5 E11antibodies was performed as described in detail above.

After 6 doses of the recombinant E11 (amino acid residues 745-850 of SEQID NO:1), serum was analyzed for FcRH5 binding antibodies using FACS.Significant reactivity was detected to SVT2 cells that express humanfull length FcRH5, cyno full length FcRH5, or the human E11 domaintransmembrane domain and cytoplasmic domains but not vector transfectedSVT2 cells indicating that FcRH5 reactive antibodies were present in thesera of all 5 immunized mice.

After 9 doses, lymphocytes from the immunized mice were electrofusedwith X63-Ag8.653 mouse myeloma cells. 323 IgG positive hybridomasubclones were selected for further screening. Clones were tested forbinding to recombinant E11 protein (amino acid residues 745-850 of SEQID NO:1) by ELISA (not shown) and binding to SVT2 cells that expresshuman full length FcRH5, cyno full length FcRH5 or human E11 domaintransmembrane domain and cytoplasmic domains of FcRH5 by FACS. A totalof 26 clones were identified that bound to cells that express humanFcRH5 and cells that express cyno FcRH5, indicative of cross-speciesreactivity (Table 2). Subclone supernatants were further characterizedfor binding to A) multiple myeloma cells transfected with human FcRH5,B) cells that express human FcRH5 endogenously (MOLP-2 myeloma cells,peripheral human CD19+ B-cells from healthy donors), C) SVT2 cellstransfected to express human FcRH1, FcRH2, FcRH3 or FcRH4, D) 293 cellsthat express truncated version of human FcRH5 (lacking 4 Ig-domainsincluding E11; amino acids 464-850 of SEQ ID NO:1) and E) NK-cells. Inaddition, binding of supernatants to soluble FcRH5a was analyzed byELISA. Based on these analysis monoclonal antibodies were selected forpurification.

TABLE 2 Sample SVT2-huFcRH5 SVT2-cyFcRH5 SVT2-huE11 1B8 +++ ++ ++ 4H8+++ ++ ++ 1H11 +++ ++ + 4G8 ++ + + 4D4 + + + 1C8 +++ ++ + 3C10 +++ ++ ++3A4 +++ ++ ++ 6D2 +++ +++ + 3G3 ++ + +++ 1F4 ++ + + 3F10 ++ + +++ 1G7+++ ++ ++ 3B12 ++ + +++ 3G7 +++ ++ + 5A10 +++ ++ ++ 1C12 ++ + + 3D12++ + ++ 5H4 ++ ++ + 5H9 ++ ++ +++ 3C5 ++ ++ + 2D10 + + +++ 5B12 ++ + ++1H2 + + + 5F1 ++ ++ ++ 2H7 ++ +++ ++

FIG. 2 shows the dose-range of binding of five purified E11 antibodies,non-isoform selective anti-FcRH5 antibody 10A8 (which binds Ig-likedomains 4-5 of FcRH5c) and a control antibody specific to the N-terminalgD-tag to the SVT2 cells expressing either human FcRH5 (FIG. 2A) or cynoFcRH5 (FIG. 2B). Antibodies in this assay were directly labeled withAPC-fluorophore according to manufacturer's protocol (Invitrogen#z25051, z25151, z25251). Binding of representative E11 antibody 5A10 tohuman FcRH5 transfected EJM (FIG. 3A) and OPM2 (FIG. 3B) multiplemyeloma cell lines was found to be similar or better compared previouslydescribed non-isoform selective FcRH5 antibodies 10A8 and 7D11 (bothbind Ig-like domains 4-5 of FcRH5c) (Elkins et al., 2012; Polson et al.,2006). MOLP-2 cells are one of the very few known multiple myeloma celllines that express low levels of FcRH5 endogenously. 5A10, 5F1, 3G7 and6D2 subclone supernatants stained MOLP-2 cells with intensity similar to7D11 (FIGS. 3C-F).

Two separate tests were designed to address dependency of binding on thepresence of membrane proximal Ig-domain 9 (E11). First a truncated humanFcRH5c mutant was generated that lacks Ig-domains 6-9 (amino acids464-850 of SEQ ID NO:1) including the expected binding site for theantibodies derived from E11 immunization. This construct with N-terminalgD-tag was expressed in 293 cells and subjected to 2.5 ug/ml subclonesupernatants followed by PE labeled goat anti-mouse secondary antibody(1:1000 dilution). None of the tested subclones bound to 293 cells thatexpress the truncated human FcRH5c (FIG. 4A). In contrast binding wasdetected to 293 cells that express wild type human FcRH5c. Binding of gDor non-isoform selective antibody clone (10A8) was not altered by themutation. This result demonstrates that binding site of the E11antibodies was included in Ig-domains 6-9.

Isoform selectivity was further demonstrated by testing binding to thesoluble FcRH5a isoform. For this, 293 cells were transfected to expressthe soluble isoform with C-terminal HIS-expression tag. Expression ofFcRH5a protein was confirmed with Western blot analysis using anti-HISantibody. A 65 kD band was detected in conditioned media from FcRH5a butnot vector transfected cells (not shown). For the ELISA, plates werecoated with anti-HIS capture antibody and incubated 1 hour with 1:10diluted conditioned media including the HIS-tagged soluble FcRH5aisoform. The E11 monoclonal antibodies were used for detection in1-0.001 ug/ml concentration, incubated for 1 hour followed by incubationwith goat anti-mouse HRP antibody and finally with TMB-substrate. Whileclones 2H7 and 5A10 demonstrate considerable reactivity to solubleFcRH5a, the other tested monoclonal antibodies do not show anydetectable binding (FIG. 5A). This result confirms that the Ig-domain 9(E11) is required for binding of the antibodies 1G7, 3A4, 3B12, 3G7 and5F1, and therefore these antibodies are selective for full the lengthFcRH5 isoform (FcRH5c).

FcRH5 is expressed endogenously in B-cells (Hatzivassiliou et al., 2001;Polson et al., 2006). To evaluate binding of subclone supernatants toB-cells, PBMCs were extracted from the blood of healthy donors. 1million human PBMC were incubated with 4 ug/ml subclone supernatants for60 min, washed and incubated with 1:100 dilution of goat anti-mousesecondary labeled with APC. Cells were then washed again twice andstained PE-labeled anti-CD19 (BD Biosciences #340364) prior flowcytometry and analysis of binding to CD19+ cells. Most of thesupernatants induced a significant shift in the APC signal in CD19+cells (FIG. 5B) over the controls (no primary antibody, anti-gD)indicative of binding to B cells.

Fc receptor homolog (FcRH) family molecules have a high degree ofhomology to one another (Miller et al., 2002). The homology isespecially high between the membrane proximal domains, which the E11antibodies target (Miller et al., 2002). To investigate the crossreactivity to family members, FcRH1, FcRH2, FcRH3 and FcRH4 (allincluding an N-terminal gD-expression tag) were expressed in SVT2 cellsand cells were stained with subclone supernatants and goat anti-mouse-PEsecondary antibody. Expression of the transfected FcRH was confirmed bya signal from anti-gD antibody in all cell lines. None of thesupernatants bound significantly to FcRH2 expressing cells as comparedto staining with the gD antibody (FIG. 6B). 1B8, 1H11, 3C10, 4G8 and 6D2demonstrated a low level of binding to FcRH1 (FIG. 6A) and 1F4 bound toFcRH4 (FIG. 6D). Overall, the signals from FcRH3-expressing SVT2 cellswere low, including the gD control antibody, indicative of lowexpression level. Low level of binding to FcRH3-expressing SVT2 cellswas detected for 1F4 and 4H8 supernatants (FIG. 6C).

Since the overall signal in the FcRH3-expressing SVT2 cells was low,further testing was done using PBMCs from healthy donors. PBMCs werestained as described above, but instead of CD19, CD56 (BD Biosciences#555516) was used to gate the investigated cell population to NK cells.NK-cells express endogenously FcRH3 (Polson et al., 2006), and asexpected, were stained by a previously described monoclonal anti-FcRH3antibody (Polson et al., 2006). FcRH1 expression was also detected inCD56+ cells, but none of the E11 subclone supernatants significantlystained the NK cells (FIG. 7) demonstrating lack of cross reactivity toendogenously expressed FcRH3.

The cross reactivity of the family members were re-tested using theidentical protocol described above in SVT2 cells but using freshreagents and re-transfecting SVT2 cells with FcRH1, FcRH2, FcRH3, andFcRH4. Re-testing the purified antibodies as described above resulted insignificantly different results than the first series of experiments.These updated results are summarized in Table 4. Rather than showinglittle to no cross-reactivity with other FcRH family members, all butone antibody (1G7) showed significant binding to both FcRH5 and at leastone or more other family members.

Without being bound by theory, this amount of antibody cross-reactivityis what would be expected, given the sequence similarity of the lastIg-like domain in the various FcRH family members.

CD8+ T cells are among the most potent immune effector cells. Theactivity of T cells can be recruited to kill tumor cells by usingbispecific antibodies (or antibody fragments) that simultaneously bindboth T cell and a tumor antigen. The dual binding can lead to apolyclonal activation of T cells and specific killing of tumor antigenexpressing cells (Liu et al., 1985; Shalaby et al., 1992). Several tumortargets and several bispecific antibody platforms have demonstratedgeneral flexibility and preclinical feasibility for this approach.Importantly, promising clinical activity has been demonstrated with aCD19 targeting, T cell activating bispecific scFv antibody fragmentblinatumomab (MT103; MicroMet). Treatment with doses as low as 60ug/m²/day results in prolonged responses in clinical trials fortreatment of relapsed non-Hodgkin's lymphoma and acute lymphoblasticleukemia (Bargou et al., 2008; Dreier et al., 2002)

The ability of the FcRH5 antibodies to activate T cell and mediatekilling in bispecific antibody format was investigated by generatingbispecific bisFab molecules. In short, these bispecific molecules aregenerated by proteolytical cleavage of the antibody, followed byreduction, re-oxidation reactions and conjugation of Fab-fragments usingbis-maleamide (Scheer et al., 2012b and as described above). Anti-CD3antibody clone UCHT1 binds to human CD3 that incorporates to T cellreceptor. UCHT1.v9 has previously been shown to be efficient T cellbinding arm (Junttila et al., 2012 and as described above; Zhu et al.,1995) and therefore was used to the FcRH5 bisFabs. Nine anti-FcRH5antibody clones (1G7, 2H7, 3G7, 5A10, 5F1, 6D2, 3B12, 3C10, 3F10) fromthe E11 immunization were chosen for the target arm and conjugated withUCHT1.v9 to result in CD3-FcRH5 bispecific bisFab molecules.

In addition to bisFab molecules, also full length bispecific antibodies(T cell dependent bispecific antibodies; TDBs) were produced usingknobs-into-holes technology (Merchant et al., 1998), which relies on apair of complementary engineered Fc regions that driveheterodimerization of antibody hemimers. As in the case of bisFabs, theUCHT1.v9 (Zhu et al., 1995) was used as the anti-CD3 (hole). For thetarget arm (knob), antibody clones from the FcRH5 E11-immunization, anon-isoform selective anti-FcRH5 clone (10A8) (Elkins et al., 2012) oranti-HER2 clone 4D5 (trastuzumab) (Carter et al., 1992) were used.Generation and purification of the TDBs has been described in detail(Junttila et al., 2012; Scheer et al., 2012a and as described above).

The ability of the bispecific molecules to mediate killing of FcRH5transfected 293 target cells was investigated by incubating the targetswith CD8+ T cells (effector cells) for 48 hours and measuring thekilling activity using Cell Titer Glo assay or FACS killing assay(assays described above). All nine bisFabs that incorporated ananti-FcRH5 E11 target arm were efficient in mediating target cellkilling (FIG. 8A-B). Killing activity was detected as low as 1-10 ng/mlconcentrations and saturated at 10-100 ng/ml concentration. Maximalkilling activity exceeded 80% for most of the clones. The killingactivity was similar compared to the HER2-TDB (FIG. 8A-B). Human HER2 isexpressed in the 293 cells on low level (data not shown). In contrast,killing activity far exceeded the non-isoform selective FcRH5-TDB(10A8), which was capable in killing only approximately 20% of thetargets (FIG. 8A-B). Similar robust activity was detected using a fulllength TDB format incorporating 2H7, 3G7 and 5A10 FcRH5-E11 clones astarget arms (FIG. 8C-D). No significant difference was detected betweenTDB and bisFab versions of 2H7 and 3G7 indicating that Fc is neithernecessary for the activity nor inhibitory for the killing activity.FcRH5 bisFabs and full length TDBs incorporating 2H7 and 3G7 as targetarm were also able to efficiently mediate killing of MOLP2 cells, whichexpress endogenously low levels of FcRH5 (FIG. 9A). T cell activationwas followed in the reactions measuring the proportion of CD8+ cellsthat express CD69 on the cell membrane. T cell activation correspondedthe killing activity and was similar for both bisFabs and TDBs (FIG.9B). A summary of the results are shown in Table 3.

TABLE 3 IHC pellet IHC Molp SVT2/ SVT2/ SVT2/ SVT2/ SVT2/ SVT2/293/huFCRH5 293/ B- Mono NK Clone cell tonsils 2 human Cyno FcRH1 FcRH2FcRH3 FcRH4 WT Mutant cell -cyte cell IRTA2a 1H2.2 − − + +/− + − − − − +− − − − − 1H2.5 − − + +/− +/− − − − − + − − − − − 1F4.1 − − +/− +/− p −− − − + − +/− − − − 1F4.2 − − + +/− +/− − − − − + − − − − − 1G7.2 − − ++++ + − − − − + − − − − − 1G7.4 − − + ++ + − − − − + − +/− − − − 1C8.1 +− + ++ ++ − − − − + − ++ − − +/− 1C8.3 + − + ++ ++ − − − − + − ++ − −+/− 2H7.3 + + + +++ +++ − +/− − − + − + − − ++ 2H7.4 + − ++ +++ +++ −+/− − − + − + − − ++ 2D10.3 + + + + +/− − − − + − +/− − − +2D10.4 + + + + +/− − − − + − + − − − 3F10.2 + − + + + − − − + − +/− − −− 3F10.7 + − + + + − − − + − + − − − 3A4.2 − − + ++ ++ − − − − + − + − −++ 3A4.4 − − ++ ++ ++ − − − − + − + − − ++ 3G7.1 − − ++ + + − − − − + −++ − − + 3B12.1 + + + ++ + − − − − + − ++ − − − 3B12.2 + + + ++ + − − −− + − ++ − − − 4G8.1 + − + ++ +/− − − − − + − + − − − 5F1.1 − − ++ ++ ++− − − − +/− − + − − − 5F1.2 − − + ++ ++ − − − − + − + − − − 5A10.1 + +++ +++ +++ − − − − + − + − − +++ 6D2.2 − − ++ ++ ++ − − − − + − + − − +3G3.5 + − + + + − − − − + − + − − − 3G3.7 + − + + + − − − − ++ − + − − −3C10.3 + + + ++ + − − − − ++ − + − − + 3C10.4 + + + ++ + − − − − ++ − +− − +

TABLE 4 SVT2/ SVT2/ SVT2/ SVT2/ Clone FcRH1 FcRH2 FcRH3 FcRH4 SVT2/FcRH51G7.2.mIgG1 − − − − +++ 2H7.3.mIgG2b − +++ +++ − +++ 3G7.1.mIgG2a − +++++ − +++ 5F1.1.mIgG2a +/− +/− +++ − +++ 5A10.1.mIgG2b − +++ +++ − +++3B12.1.mIgG2b − − +++ − +++ 3A4.2.hIgG1 − ++ +++ − +++ 6D2.2.hIgG1 − +++++ − +++ 1C8.1.hIgG1 − ++ +++ − +++ 3C10.3.hIgG1 +++ +/− − − +++3F10.7.hIgG1 − − ++ − ++

REFERENCES

-   Bargou, R. et al. (2008). Science 321, 974-977.-   Carter, P. et al. (1992). Proc Natl Acad Sci USA 89, 4285-4289.-   Dreier, T. et al. (2002). Int J Cancer 100, 690-697.-   Elkins, K. et al. (2012). Mol Cancer Ther 11, 2222-2232.-   Hatzivassiliou, G. et al. (2001). Immunity 14, 277-289.-   Liu, M. A. et al. (1985). Proc Natl Acad Sci USA 82, 8648-8652.-   Merchant, A. M. et al. (1998). Nat Biotechnol 16, 677-681.-   Miller, I et al. (2002). Blood 99, 2662-2669.-   Polson, A. G. et al. (2006). Expression pattern of the human    FcRH/IRTA receptors in normal tissue and in B-chronic lymphocytic    leukemia. International immunology 18, 1363-1373.-   Shalaby, M. R. et al. (1992). J Exp Med 175, 217-225.-   Zhu, Z. et al. (1995). Int J Cancer 62, 319-324.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention. The disclosures of all patent andscientific literature cited herein are expressly incorporated in theirentirety by reference.

Variable Light Chain Domain 1C8.1 (SEQ ID NO: 110)DIVMTQSQRFMSTSLGDRVSVTCKASQNVITNVAWYQQKPGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFCQQYTNYPMWTFGGGTRLEIKRTVA 1G7.2 (SEQ ID NO: 112)DIVMTQSHKIMSTSVGDRVSITCKASQDVSNIVVWFQQKPGQSPNLLIYSASYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQQHYSSPYTFGGGTKLEIKRTVAA 2H7.3 (SEQ ID NO: 114)EIVLTQSPATLSVTPGDSVSLSCRASQNIRNNLHWYQQKSHESPRLLIKFTSQSISGIPSRFTGSGSGTDFTLSINSVETEDFGMYFCQQSNNWPQYTFGGGTKLEIKRTVAA  3A4.2 (SEQ ID NO: 116)DIQMTQSPATLSVTPGDSVSLSCRASQSISNNLHWYQQKSHESPRLLIKFASQSISGIPSRFSGSGSGTDFTLSINSVETEDFGMYFCQQSNNWPQYTFGGGTKLELKRTVAA 3B12.1.1 (SEQ ID NO: 118)DIQMTQSPASLSASVGETVTITCRASENIYSNLAWYQLKQGKSPQLLVYGAANLAEGVPSRISGSGSGTQYSLKINSLQSEDFGTYYCQHFWGIPWTFGGGTKLEIKRTVAA 3C10 (SEQ ID NO: 120)DIQMTQTPLSLPVTLGDQASISCRSSQSLVHRNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPPTFGGGTKLELKRTVAA 3F10 (SEQ ID NO: 122)DIVMTQSPASLSASVGETVTITCRASENIYSNLAWYQLKQGKSPQLLVYGAANLAEGVPSRISGSGSGTQYSLKINSLQSEDFGTYYCQHFWGIPWTFGGGTKLEIKRTVAA  3G3 (SEQ ID NO: 124)DIVMTQSPASLSASVGETVTITCRASENIYSNLAWYQLKQGKSPQLLVYGAANLAEGVPSRISGSGSGTQYSLKINSLQSEDFGTYYCQHFWGIPWTFGGGTKLEIKRTVAA  3G7.1.5 (SEQ ID NO: 126)DIVLIQSPATLSVTLGGSVSLSCRASQSISNNLHWYQQKSHESPRLLIKFASQSISGIPSRFRGSGSGTDFTLTINSVETEDFGIYFCQQSNNWPQYTFGGGTKLELKRTVAA 5A10.1.3 (SEQ ID NO: 128)DIVLTQSPANLSVIPGDSVSLSCRASQNIRNNLHWYQQKSQESPRLLIKFASQSMSGTPSRFTGSGSGTDFTLTINTVETEDFGMYFCQQSNNWPQYTFGGGTKLEIKRTVAA  5F1.1.5 (SEQ ID NO: 130)QAVVTQESALTTSPGETVTLTCRSSTGTVTTSNFANWVQEKPDHLFTGLIGGTSNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCVLWCSNLWVFGGGTKLTVLGQPKAA 6D2 (SEQ ID NO: 132)DIVMTQSHKFMSTSVGDRVSITCKASQDVGTAVAWYQQKPGQSPKLLIFWPSTRHTGVPDRFTGSGSGTDFTLTIGNVQSEDLADYFCQQFSSLPHTFGGGTKLEIKRTVAA  1G7′ (SEQ ID NO: 134)DIVMTQSHKIMSTSVGDRVSITCKASQDVSNIVVWFQQKPGQSPNLLIYSASYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQQHYSSPYTFGGGTKLEIK  Variable Heavy Chain Domain 1C8.1(SEQ ID NO: 111)EVQLQQSGPELVKPGASMKISCEASGYSFTAYIMNWVKQSRGKNLEWIGLINPYNGETTYNQKFKGKATLTVDQSSSTAYMELLSLTSEDSAVYFCARGLYWFPYWGQGTLVTVSAASTKGPSVFPLAP  1G7.2(SEQ ID NO: 113)EVQLQESGPGLVQPSQSLSITCTVSGFSLTRFGVHWVRQSPGKGLEWLGVIWRGGSTDYNAAFMSRLTITKDNSKSQVFFKLNSLKVDDTAIYYCSNHYYGSSDYALDNWGQGTSVTVSSASTKGPSVFPLAP  2H7.3(SEQ ID NO: 115)EVQLQQSGPELWKPGASVKMSCKASGYTFTDYYMKWVKQTHGKSLEWIGDINPNNGETFYSQKFKGKATLTVDKSSTTAYMQLNSLTSEDSAVYYCARGLYRFDYWGQGTTLTVSSASTKGPSVFPLAP  3A4.2(SEQ ID NO: 117)EVQLQQSGPELVKSGASVKMSCKASGYTFTDYYMKWVKQSHGKSLEWIGDINPYNGETFYNQKLKGKATLTVDKSSNTVFMQLNSLTSEDSAVYYCARGLYFFAYWGQGTTLTVSSASTKGPSVFPLAP 3B12.1.1(SEQ ID NO: 119)EVQLQQSGPELVKPGASVKISCKTSGYTFTEYTIHWVKQSHGKSLERIGGINPNNDAVSYNQRFRGKATLTVDKSSSTAYMELRSLTSEDSAVYYCAKLGRGYYFDYWGQGTTLTVSSASTKGPSVFPLAP  3C10 (SEQ ID NO: 121)QVQLQQPGAELVRPGASVKLSCKTSGYTFISYWINWVKQRPGQGLEWIGNIYPSDSYTNYNQKFKDKATLTVDTSSSTAYMQLTSPTSEDSAVYYCTRSLYGYDASYFDYWGQGTTLTVSSASTKGPSVFPLAP  3F10(SEQ ID NO: 123)QVQLQQSGPELVKPGASVKISCKTSGYTFTEYTIHWVKQSHGKSLERIGGINPNNDAISYNQKFRGKATLTVDKSSSTAYMELRSLTSEDSAVYYCAKLGRGYYFDYWGRGTTLTVSSASTKGPSVFPLAP  3G3 (SEQ ID NO: 125)EVQLQQSGPELVKPGASVKISCKTSGYTFTEYTIHWVKQSHGKSLERIGGINPNNDAISYNQKFRGKATLTVDKSSSTAYMELRSLTSEDSAVYYCAKLGRGYYFDYWGRGTTLTVSSASTKGPSVFPLAP  3G7.1.5(SEQ ID NO: 127)EVQLQQSGPELVKPGASVKMSCKASGYTFTDYYMKWVRQNHGKRLEWIGDINPYNGDTFYNQKFKDKATLTVDKSSSTAYMQFNSLTSEDSAVYYCARGLYFFHYWGQGTTLTVSSASTKGPSVFPLAP  5A10.1.3(SEQ ID NO: 129)EVQLQQSGPELWKPGASVKMSCKASGYTFTDYYMKWVKQSHGKSLEWIGDINPNNGETFYNQKFKGKATLTVDKSTSTAYMELNSLTTEDSAVYYCARGLYRFDYWGQGTTLTVSSAASTKGPSVFPLAP  5F1.1.5(SEQ ID NO: 131)QVQLQQSGADLVRPGTSVKVSCKASGYAFTNYLIEWVKQRPGQGLEWIGVINPGSGGTNYNEKFKGKATLTADKSSSTAYMQLSSLTSDDSAVYFCARTRNYGYVIDYWGQGTTLTVSSASTKGPSVFPLAP 6D2(SEQ ID NO: 133)QVQLQQSGPELVKPGASVKISCKASGFSFTAYFMNWVKQSHGKSPEWIGRINPYNGETFFNQNFKDKATLTVDKSSNTAHMELLSLTSDDSAVYYCGRGLYYLNYWGQGTTLTVSSASTKGPSVFPLAP 1G7′ (SEQ ID NO: 135)QVQLKQSGPGLVQPSQSLSITCTVSGFSLTRFGVHWVRQSPGKGLEWLGVIWRGGSTDYNAAFMSRLTITKDNSKSQVFFKLNSLKVDDTAIYYCSNHYYGSSDYALDNWGQGISVTVSS 

Kabat CDR L1 (HVR-L1)

24 25 26 27 27A 27B 27C 27D 27E 27F 28 29 30 31 32 33 34 SEQ ID NO:1C8.1 K A S Q . . . . . . N V I T N V A 2 1G7.2 K A S Q . . . . . . D VS N I V V 3 2H7.3 R A S Q . . . . . . N I R N N L H 4 3A4.2 R A S Q . .. . . . S I S N N L H 5 3812.1.1 R A S E . . . . . . N I Y S N L A 63C10 R S S Q S L V H R . N G N T Y L H 7 3F10 R A S E . . . . . . N I YS N L A 8 3G3 R A S E . . . . . . N I Y S N L A 9 3G7.1.5 R A S Q . . .. . . S I S N N L H 10 5A10.1.3 R A S Q . . . . . . N I R N N L H 115F1.1.5 R S S T G T V . . . T T S N F A N 12 6D2 K A S Q . . . . . . D VG T A V A 13

Kabat CDR L2 (HVR-L2)

50 51 52 53 54 54A 54B 54C 54D 54E 55 56 SEQ ID NO: 1C8.1 S A S Y R . .. . . Y S 14 1G7.2 S A S Y R . . . . . Y T 15 2H7.3 F T S Q S . . . . .I S 16 3A4.2 F A S Q S . . . . . I S 17 3B12.1.1 G A A N L . . . . . A E18 3C10 K V S N R . . . . . F S 19 3F10 G A A N L . . . . . A E 20 3G3 GA A N L . . . . . A E 21 3G7.1.5 F A S Q S . . . . . I S 22 5A10.1.3 F AS Q S . . . . . M S 23 5F1.1.5 G T S N R . . . . . A P 24 6D2 W P S T R. . . . . H T 25

Kabat CDR L3 (HVR-L3)

89 90 91 92 93 94 95 95A 95B 95C 95D 95E 95F 96 97 SEQ ID NO: 1C8.1 Q QY T N Y Y P M . . . . W T 26 167.2 Q Q H Y S P . . . . . . . Y T 272H7.3 Q Q S N N W P Q . . . . . Y T 28 3A4.2 Q Q S N N W P Q . . . . . YT 29 3812.1.1 Q H F W G I P . . . . . . W T 30 3C10 S Q S T H V P . . .. . . P T 31 3F10 Q H F W G I P . . . . . . W T 32 3G3 Q H F W G I P . .. . . . W T 33 3G7.1.5 Q Q S N N W P Q . . . . . Y T 34 5A10.1.3 Q Q S NN W P Q . . . . . Y T 35 5F1.1.5 V L W C S N L . . . . . . W V 36 6D2 QQ F S S L P . . . . . . H T 37

Kabat CDR H1 (HVR-H1)

31 32 33 34 35 35A 35B SEQ ID NO 1C8.1 A Y I M N . . 38 1G7.2 R F G V H. . 39 2H7.3 D Y Y M K . . 40 3A4.2 D Y Y M K . . 41 3B12.1.1 E Y T I H. . 42 3C10 S Y W I N . . 43 3F10 E Y T I H . . 44 3G3 E Y T I H . . 453G7.1.5 D Y Y M K . . 46 5A10.1.3 D Y Y M K . . 47 5F1.1.5 N Y L I E . .48 6D2 A Y F M N . . 49

CDR H1 (HVR-H1)

SEQ ID 26 27 28 29 30 31 32 33 34 35 35A 35B NO: 1C8.1 G Y S F T A Y I MN . . 50 1G7.2 G F S L T R F G V H . . 51 2H7.3 G Y T F T D Y Y M K . .52 3A4.2 G Y T F T D Y Y M K . . 53 3612.1.1 G Y T F T E Y T I H . . 543C10 G Y T F I S Y W I N . . 55 3F10 G Y T F T E Y T I H . . 56 3G3 G YT F T E Y T I H . . 57 3G7.1.5 G Y T F T D Y Y M K . . 58 5A10.1.3 G Y TF T D Y Y M K . . 59 5F1.1.5 G Y A F T N Y L I E . . 60 6D2 G F S F T AY F M N . . 61 SEQ 50 51 52 52A 52B 52C 53 54 55 56 57 58 59 60 61 62 6364 65 ID NO 1C8.1 L I N P . . Y N G E T T Y N Q K F K G 62 1G7.2 V I W .. . R G G S T D Y N A A F M S 63 2H73 D I N P . . N N G E T F Y S Q K FK G 64 3A42 D I N P . . Y N G E T F Y N Q K L K G 65 3B12.1.1 G I N P .. N N D A V S Y N Q R F R G 66 3C10 N I Y P . . S D S Y T N Y N Q K F KD 67 3F10 G I N P . . N N D A I S Y N Q K F R G 68 3G3 G I N P . . N N DA I S Y N Q K F R G 69 3G7.1.5 D I N P . . Y N G D T F Y N Q K F K D 705A101.3 D I N P . . N N G E T F Y N Q K F K G 71 5F1.1.5 V I N P . . G SG G T N Y N E K F K G 72 6D2 R I N P . . Y N G E T F F N F N F K D 73

Kabat CDR H2 (HVR-H2)

49 50 51 52 52A 52B 52C 53 54 55 56 57 58 59 60 61 62 63 64 65 SEQ IDNO: 1C8.1 G L I N P . . Y N G E T T Y N Q K F K G 74 1G7.2 G V I W . . .R G G S T D Y N A A F M S 75 2H73 G D I N P . . N N G E T F Y S Q K F KG 76 3A42 G D I N P . . Y N G E T F Y N Q K L K G 77 3B12.1.1 G G I N P. . N N D A V S Y N Q R F R G 78 3C10 G N I Y P . . S D S Y T N Y N Q KF K D 79 3F10 G G I N P . . N N D A I S Y N Q K F R G 80 3G3 G G I N P .. N N D A I S Y N Q K F R G 81 3G7.1.5 G D I N P . . Y N G D T F Y N Q KF K D 82 5A101.3 G D I N P . . N N G E T F Y N Q K F K G 83 5F1.1.5 G VI N P . . G S G G T N Y N E K F K G 84 6D2 G R I N P . . Y N G E T F F NQ N F K D 85

Kabat CDR H3 (HVR-H3)

SEQ 100 100 100 100 100 100 100 100 100 100 100 100 100 ID 95 96 97 9899 100 A B C D E F G H I J K L M 101 102 NO: 1C8.1 G L Y . . . . . . . .. . . . . . W F P Y 86 1G7.2 H Y Y G S S D . . . . . . . . . Y A L D N87 2H73 G L Y R . . . . . . . . . . . . . . F D Y 88 3A42 G L Y F . . .. . . . . . . . . . . F A Y 89 3B12.1.1 L G R G Y . . . . . . . . . . .. Y F D Y 90 3C10 S L Y G Y D A S . . . . . . . . . Y F D Y 91 3F10 L GR G Y . . . . . . . . . . . . Y F D Y 92 3G3 L G R G Y . . . . . . . . .. . . Y F D Y 93 3G7.1.5 G L Y F . . . . . . . . . . . . . . F H Y 94

CDR H3 (HVR-H3)

SEQ 100 100 100 100 100 100 100 100 100 100 100 100 100 ID 93 94 95 9697 98 99 100 A B C D E F G H I J K L M 101 102 NO: 1C8.1 A R G L Y . . .. . . . . . . . . . . W F P Y 98 1G7.2 S N H Y Y G S S D . . . . . . . .. Y A L D N 99 2H73 A R G L Y R . . . . . . . . . . . . . . F D Y 1003A42 A R G L Y F . . . . . . . . . . . . . . F A Y 101 3B12.1.1 A K L GR G Y . . . . . . . . . . . . Y F D Y 102 3C10 T R S L Y G Y D A S . . .. . . . . . Y F D Y 103 3F10 A K L G R G Y . . . . . . . . . . . . Y F DY 104 3G3 A K L G R G Y . . . . . . . . . . . . Y F D Y 105 3G7.1.5 A RG L Y F . . . . . . . . . . . . . . F H Y 106 5A101.3 A R T R N Y G Y V. . . . . . . . . . . F D Y 107 5F1.1.5 A R T R N Y G Y V . . . . . . .. . . . I D Y 108 6D2 G R G L Y . . . . . . . . . . . . . . Y L N Y 109

FcRH5c (SEQ ID NO: 1)MLLWVILLVLAPVSGQFARTPRPIIFLQPPWTTVFQGERVTLTCKGFRFYSPQKTKWYHRYLGKEILRETPDNILEVQESGEYRCQAQGSPLSSPVHLDFSSASLILQAPLSVFEGDSVVLRCRAKAEVTLNNTIYKNDNVLAFLNKRTDFHIPHACLKDNGAYRCTGYKESCCPVSSNTVKIQVQEPFTRPVLRASSFQPISGNPVTLTCETQLSLERSDVPLRFRFFRDDQTLGLGWSLSPNFQITAMWSKDSGFYWCKAATMPYSVISDSPRSWIQVQIPASHPVLTLSPEKALNFEGTKVTLHCETQEDSLRTLYRFYHEGVPLRHKSVRCERGASISFSLTTENSGNYYCTADNGLGAKPSKAVSLSVTVPVSHPVLNLSSPEDLIFEGAKVTLHCEAQRGSLPILYQFHHEGAALERRSANSAGGVAISFSLTAEHSGNYYCTADNGFGPQRSKAVSLSVTVPVSHPVLTLSSAEALTFEGATVTLHCEVQRGSPQILYQFYHEDMPLWSSSTPSVGRVSFSFSLTEGHSGNYYCTADNGFGPQRSEVVSLFVTVPVSRPILTLRVPRAQAVVGDLLELHCEAPRGSPPILYWFYHEDVTLGSSSAPSGGEASFNLSLTAEHSGNYSCEANNGLVAQHSDTISLSVIVPVSRPILTFRAPRAQAVVGDLLELHCEALRGSSPILYWFYHEDVTLGKISAPSGGGASFNLSLTTEHSGIYSCEADNGLEAQRSEMVTLKVAVPVSRPVLTLRAPGTHAAVGDLLELHCEALRGSPLILYRFFHEDVTLGNRSSPSGGASLNLSLTAEHSGNYSCEADNGLGAQRSETVTLYITGLTANRSGPFATGVAGGLLSIAGLAAGALLLYCWLSRKAGRKPASDPARSPSDSDSQEPTYHNVPAWEELQPVYTNANPRGENVVYSEVRIIQEKKKHAVASDPRHLRNKGSPIIYSEVKVASTPVSGSLFLASSAPHR

1. An isolated anti-FcRH5 antibody that binds an isoform c-specificregion of the extracellular domain of FcRH5c.
 2. The antibody of claim1, wherein the isoform c-specific region comprises Ig-like domain
 9. 3.The antibody of claim 1, wherein the isoform c-specific region comprisesamino acids 743-850 of SEQ ID NO:1.
 4. The antibody of claim 1, whereinthe antibody comprises: a) a heavy chain comprising a HVR-H1 comprisingthe amino acid sequence of SEQ ID NO:38, HVR-H2 comprising the aminoacid sequence of SEQ ID NO:62, and HVR-H3 comprising the amino acidsequence of SEQ ID NO:86; and/or b) a light chain comprising a HVR-L1comprising the amino acid sequence of SEQ ID NO:2, HVR-L2 comprising theamino acid sequence of SEQ ID NO:14, and HVR-L3 comprising the aminoacid sequence of SEQ ID NO:26.
 5. The antibody of claim 4, wherein theheavy chain comprises a HVR-H1 comprising the amino acid sequence of SEQID NO:50, HVR-H2 comprising the amino acid sequence of SEQ ID NO:74, andHVR-H3 comprising the amino acid sequence of SEQ ID NO:98.
 6. Theantibody of claim 1, wherein the antibody comprises: a) a heavy chaincomprising a HVR-H1 comprising the amino acid sequence of SEQ ID NO:39,HVR-H2 comprising the amino acid sequence of SEQ ID NO:63, and HVR-H3comprising the amino acid sequence of SEQ ID NO:87; and/or b) a lightchain comprising a HVR-L1 comprising the amino acid sequence of SEQ IDNO:3, HVR-L2 comprising the amino acid sequence of SEQ ID NO:15, andHVR-L3 comprising the amino acid sequence of SEQ ID NO:27.
 7. Theantibody of claim 6, wherein the heavy chain comprises a HVR-H1comprising the amino acid sequence of SEQ ID NO:51, HVR-H2 comprisingthe amino acid sequence of SEQ ID NO:75, and HVR-H3 comprising the aminoacid sequence of SEQ ID NO:99.
 8. The antibody of claim 1, wherein theantibody comprises: a) a heavy chain comprising a HVR-H1 comprising theamino acid sequence of SEQ ID NO:40, HVR-H2 comprising the amino acidsequence of SEQ ID NO:64, and HVR-H3 comprising the amino acid sequenceof SEQ ID NO:88; and/or b) a light chain comprising a HVR-L1 comprisingthe amino acid sequence of SEQ ID NO:4, HVR-L2 comprising the amino acidsequence of SEQ ID NO:16, and HVR-L3 comprising the amino acid sequenceof SEQ ID NO:28.
 9. The antibody of claim 8, wherein the heavy chaincomprises a HVR-H1 comprising the amino acid sequence of SEQ ID NO:52,HVR-H2 comprising the amino acid sequence of SEQ ID NO:76, and HVR-H3comprising the amino acid sequence of SEQ ID NO:100.
 10. The antibody ofclaim 1, wherein the antibody comprises: a) a heavy chain comprising aHVR-H1 comprising the amino acid sequence of SEQ ID NO:41, HVR-H2comprising the amino acid sequence of SEQ ID NO:65, and HVR-H3comprising the amino acid sequence of SEQ ID NO:89; and/or b) a lightchain comprising a HVR-L1 comprising the amino acid sequence of SEQ IDNO:5, HVR-L2 comprising the amino acid sequence of SEQ ID NO:17, andHVR-L3 comprising the amino acid sequence of SEQ ID NO:29.
 11. Theantibody of claim 10, wherein the heavy chain comprises a HVR-H1comprising the amino acid sequence of SEQ ID NO:53, HVR-H2 comprisingthe amino acid sequence of SEQ ID NO:77, and HVR-H3 comprising the aminoacid sequence of SEQ ID NO:101.
 12. The antibody of claim 1, wherein theantibody comprises: a) a heavy chain comprising a HVR-H1 comprising theamino acid sequence of SEQ ID NO:42, HVR-H2 comprising the amino acidsequence of SEQ ID NO:66, and HVR-H3 comprising the amino acid sequenceof SEQ ID NO:90; and/or b) a light chain comprising a HVR-L1 comprisingthe amino acid sequence of SEQ ID NO:6, HVR-L2 comprising the amino acidsequence of SEQ ID NO:18, and HVR-L3 comprising the amino acid sequenceof SEQ ID NO:30.
 13. The antibody of claim 12, wherein the heavy chaincomprises a HVR-H1 comprising the amino acid sequence of SEQ ID NO:54,HVR-H2 comprising the amino acid sequence of SEQ ID NO:78, and HVR-H3comprising the amino acid sequence of SEQ ID NO:102.
 14. The antibody ofclaim 1, wherein the antibody comprises: a) a heavy chain comprising aHVR-H1 comprising the amino acid sequence of SEQ ID NO:43, HVR-H2comprising the amino acid sequence of SEQ ID NO:67, and HVR-H3comprising the amino acid sequence of SEQ ID NO:91; and/or b) a lightchain comprising a HVR-L1 comprising the amino acid sequence of SEQ IDNO:7, HVR-L2 comprising the amino acid sequence of SEQ ID NO:19, andHVR-L3 comprising the amino acid sequence of SEQ ID NO:31.
 15. Theantibody of claim 14, wherein the heavy chain comprises a HVR-H1comprising the amino acid sequence of SEQ ID NO:55, HVR-H2 comprisingthe amino acid sequence of SEQ ID NO:79, and HVR-H3 comprising the aminoacid sequence of SEQ ID NO:103.
 16. The antibody of claim 1, wherein theantibody comprises: a) a heavy chain comprising a HVR-H1 comprising theamino acid sequence of SEQ ID NO:44, HVR-H2 comprising the amino acidsequence of SEQ ID NO:68, and HVR-H3 comprising the amino acid sequenceof SEQ ID NO:92; and/or b) a light chain comprising a HVR-L1 comprisingthe amino acid sequence of SEQ ID NO:8, HVR-L2 comprising the amino acidsequence of SEQ ID NO:20, and HVR-L3 comprising the amino acid sequenceof SEQ ID NO:32.
 17. The antibody of claim 16, wherein the heavy chaincomprises a HVR-H1 comprising the amino acid sequence of SEQ ID NO:56,HVR-H2 comprising the amino acid sequence of SEQ ID NO:80, and HVR-H3comprising the amino acid sequence of SEQ ID NO:104.
 18. The antibody ofclaim 1, wherein the antibody comprises: a) a heavy chain comprising aHVR-H1 comprising the amino acid sequence of SEQ ID NO:45, HVR-H2comprising the amino acid sequence of SEQ ID NO:69, and HVR-H3comprising the amino acid sequence of SEQ ID NO:93; and/or b) a lightchain comprising a HVR-L1 comprising the amino acid sequence of SEQ IDNO:9, HVR-L2 comprising the amino acid sequence of SEQ ID NO:21, andHVR-L3 comprising the amino acid sequence of SEQ ID NO:33.
 19. Theantibody of claim 18, wherein the heavy chain comprises a HVR-H1comprising the amino acid sequence of SEQ ID NO:57, HVR-H2 comprisingthe amino acid sequence of SEQ ID NO:81, and HVR-H3 comprising the aminoacid sequence of SEQ ID NO:105.
 20. The antibody of claim 1, wherein theantibody comprises: a) a heavy chain comprising a HVR-H1 comprising theamino acid sequence of SEQ ID NO:46, HVR-H2 comprising the amino acidsequence of SEQ ID NO:70, and HVR-H3 comprising the amino acid sequenceof SEQ ID NO:94; and/or b) a light chain comprising a HVR-L1 comprisingthe amino acid sequence of SEQ ID NO:10, HVR-L2 comprising the aminoacid sequence of SEQ ID NO:22, and HVR-L3 comprising the amino acidsequence of SEQ ID NO:34.
 21. The antibody of claim 20, wherein theheavy chain comprises a HVR-H1 comprising the amino acid sequence of SEQID NO:58, HVR-H2 comprising the amino acid sequence of SEQ ID NO:82, andHVR-H3 comprising the amino acid sequence of SEQ ID NO:106.
 22. Theantibody of claim 1, wherein the antibody comprises: a) a heavy chaincomprising a HVR-H1 comprising the amino acid sequence of SEQ ID NO:47,HVR-H2 comprising the amino acid sequence of SEQ ID NO:71, and HVR-H3comprising the amino acid sequence of SEQ ID NO:95; and/or b) a lightchain comprising a HVR-L1 comprising the amino acid sequence of SEQ IDNO:11, HVR-L2 comprising the amino acid sequence of SEQ ID NO:23, andHVR-L3 comprising the amino acid sequence of SEQ ID NO:35.
 23. Theantibody of claim 22, wherein the heavy chain comprises a HVR-H1comprising the amino acid sequence of SEQ ID NO:59, HVR-H2 comprisingthe amino acid sequence of SEQ ID NO:83, and HVR-H3 comprising the aminoacid sequence of SEQ ID NO:107.
 24. The antibody of claim 1, wherein theantibody comprises: a) a heavy chain comprising a HVR-H1 comprising theamino acid sequence of SEQ ID NO:48, HVR-H2 comprising the amino acidsequence of SEQ ID NO:72, and HVR-H3 comprising the amino acid sequenceof SEQ ID NO:96; and/or b) a light chain comprising a HVR-L1 comprisingthe amino acid sequence of SEQ ID NO:12, HVR-L2 comprising the aminoacid sequence of SEQ ID NO:24, and HVR-L3 comprising the amino acidsequence of SEQ ID NO:36.
 25. The antibody of claim 24, wherein theheavy chain comprises a HVR-H1 comprising the amino acid sequence of SEQID NO:60, HVR-H2 comprising the amino acid sequence of SEQ ID NO:84, andHVR-H3 comprising the amino acid sequence of SEQ ID NO:108.
 26. Theantibody of claim 1, wherein the antibody comprises: a) a heavy chaincomprising a HVR-H1 comprising the amino acid sequence of SEQ ID NO:49,HVR-H2 comprising the amino acid sequence of SEQ ID NO:73, and HVR-H3comprising the amino acid sequence of SEQ ID NO:97; and/or b) a lightchain comprising a HVR-L1 comprising the amino acid sequence of SEQ IDNO:13, HVR-L2 comprising the amino acid sequence of SEQ ID NO:25, andHVR-L3 comprising the amino acid sequence of SEQ ID NO:37.
 27. Theantibody of claim 26, wherein the heavy chain comprises a HVR-H1comprising the amino acid sequence of SEQ ID NO:61, HVR-H2 comprisingthe amino acid sequence of SEQ ID NO:85, and HVR-H3 comprising the aminoacid sequence of SEQ ID NO:109.
 28. The antibody of claim 1, wherein theantibody comprises: a) a VH sequence having at least 95% sequenceidentity to the amino acid sequence of SEQ ID NO:111 and/or a VLsequence having at least 95% sequence identity to the amino acidsequence of SEQ ID NO: 110; b) a VH sequence having at least 95%sequence identity to the amino acid sequence of SEQ ID NO:113 and/or aVL sequence having at least 95% sequence identity to the amino acidsequence of SEQ ID NO:112; c) a VH sequence having at least 95% sequenceidentity to the amino acid sequence of SEQ ID NO:115 and/or a VLsequence having at least 95% sequence identity to the amino acidsequence of SEQ ID NO:114; d) a VH sequence having at least 95% sequenceidentity to the amino acid sequence of SEQ ID NO:117 and/or a VLsequence having at least 95% sequence identity to the amino acidsequence of SEQ ID NO:116; e) a VH sequence having at least 95% sequenceidentity to the amino acid sequence of SEQ ID NO:119 and/or a VLsequence having at least 95% sequence identity to the amino acidsequence of SEQ ID NO:118; f) a VH sequence having at least 95% sequenceidentity to the amino acid sequence of SEQ ID NO:121 and/or a VLsequence having at least 95% sequence identity to the amino acidsequence of SEQ ID NO:120; g) a VH sequence having at least 95% sequenceidentity to the amino acid sequence of SEQ ID NO:123 and/or a VLsequence having at least 95% sequence identity to the amino acidsequence of SEQ ID NO:122; h) a VH sequence having at least 95% sequenceidentity to the amino acid sequence of SEQ ID NO:125 and/or a VLsequence having at least 95% sequence identity to the amino acidsequence of SEQ ID NO:124; i) a VH sequence having at least 95% sequenceidentity to the amino acid sequence of SEQ ID NO:127 and/or a VLsequence having at least 95% sequence identity to the amino acidsequence of SEQ ID NO: 126; j) a VH sequence having at least 95%sequence identity to the amino acid sequence of SEQ ID NO:129 and/or aVL sequence having at least 95% sequence identity to the amino acidsequence of SEQ ID NO:128; k) a VH sequence having at least 95% sequenceidentity to the amino acid sequence of SEQ ID NO:131 and/or a VLsequence having at least 95% sequence identity to the amino acidsequence of SEQ ID NO:130; l) a VH sequence having at least 95% sequenceidentity to the amino acid sequence of SEQ ID NO:133 and/or a VLsequence having at least 95% sequence identity to the amino acidsequence of SEQ ID NO:132; or m) a VH sequence having at least 95%sequence identity to the amino acid sequence of SEQ ID NO:135 and/or aVL sequence having at least 95% sequence identity to the amino acidsequence of SEQ ID NO:134.
 29. The antibody of claim 28, wherein theantibody comprises: a) a VH sequence of SEQ ID NO:111 and/or a VLsequence of SEQ ID NO:110; b) a VH sequence of SEQ ID NO:113 and/or a VLsequence of SEQ ID NO:112; c) a VH sequence of SEQ ID NO:115 and/or a VLsequence of SEQ ID NO:114; d) a VH sequence of SEQ ID NO:117 and/or a VLsequence of SEQ ID NO:116; e) a VH sequence of SEQ ID NO:119 and/or a VLsequence of SEQ ID NO:118; f) a VH sequence of SEQ ID NO:121 and/or a VLsequence of SEQ ID NO:120; g) a VH sequence of SEQ ID NO:123 and/or a VLsequence of SEQ ID NO:122; h) a VH sequence of SEQ ID NO:125 and/or a VLsequence of SEQ ID NO:124; i) a VH sequence of SEQ ID NO:127 and/or a VLsequence of SEQ ID NO:126; j) a VH sequence of SEQ ID NO:129 and/or a VLsequence of SEQ ID NO:128; k) a VH sequence of SEQ ID NO:131 and/or a VLsequence of SEQ ID NO:130; l) a VH sequence of SEQ ID NO:133 and/or a VLsequence of SEQ ID NO:132; or m) a VH sequence of SEQ ID NO:135 and/or aVL sequence of SEQ ID NO:134.
 30. The antibody of claim 1, wherein theantibody is a monoclonal antibody.
 31. The antibody of claim 1, whereinthe antibody is a human, humanized, or chimeric antibody.
 32. Theantibody of claim 1, wherein the antibody is an antibody fragment thatbinds FcRH5.
 33. The antibody of claim 1, wherein the antibody is anIgG1, IgG2a or IgG2b antibody.
 34. The antibody of claim 1, wherein theantibody has one or more of the following characteristics: a) crossreactive with full length human and cyno FcRH5, b) does not cross reactwith FcRH1, FcRH2, FcRH3, and/or FcRH4, c) binds to endogenous FcRH5, d)does not cross react with FcRH5a, and e) does not cross react withanother Ig-like domain of FcRH5.
 35. The antibody of claim 1, whereinthe antibody is a bispecific antibody.
 36. The antibody of claim 35,wherein bispecific antibody binds FcRH5 and CD3.
 37. An isolated nucleicacid encoding the antibody of claim
 1. 38. A host cell comprising thenucleic acid of claim
 37. 39. A method of producing an antibodycomprising culturing the host cell of claim 38 so that the antibody isproduced.
 40. An immunoconjugate comprising the antibody of claim 1 anda cytotoxic agent.
 41. The immunoconjugate of claim 40 having theformula Ab-(L-D)p, wherein: (a) Ab is an isolated anti-FcRH5 antibodythat binds an isoform c-specific region of the extracellular domain ofFcRH5c; (b) L is a linker; (c) D is a drug selected from a maytansinoid,an auristatin, a calicheamicin, a pyrrolobenzodiazepine, and anemorubicin derivative; and (d) p ranges from 1-8.
 42. Theimmunoconjugate of claim 41, wherein D is an auristatin.
 43. Theimmunoconjugate of claim 42, wherein D has formula D_(E)

and wherein R² and R⁶ are each methyl, R³ and R⁴ are each isopropyl, R⁵is H, R⁷ is sec-butyl, each R⁸ is independently selected from CH₃,O—CH₃, OH, and H; R⁹ is H; and R¹⁸ is —C(R⁸)₂—C(R⁸)₂-aryl.
 44. Theimmunoconjugate of claim 41, wherein the drug is MMAE.
 45. Theimmunoconjugate of claim 41, wherein D is a pyrrolobenzodiazepine ofFormula A:

wherein the dotted lines indicate the optional presence of a double bondbetween C1 and C2 or C2 and C3; R² is independently selected from H, OH,═O, ═CH₂, CN, R, OR, ═CH—R^(D), ═C(R^(D))₂, O—SO₂—R, CO₂R and COR, andoptionally further selected from halo or dihalo, wherein R^(D) isindependently selected from R, CO₂R, COR, CHO, CO₂H, and halo; R⁶ and R⁹are independently selected from H, R, OH, OR, SH, SR, NH₂, NHR, NRR′,NO₂, Me₃Sn and halo; R⁷ is independently selected from H, R, OH, OR, SH,SR, NH₂, NHR, NRR′, NO₂, Me₃Sn and halo; Q is independently selectedfrom O, S and NH; R¹¹ is either H, or R or, where Q is O, SO₃M, where Mis a metal cation; R and R′ are each independently selected fromoptionally substituted C₁₋₈ alkyl, C₃₋₈ heterocyclyl and C₅₋₂₀ arylgroups, and optionally in relation to the group NRR′, R and R′ togetherwith the nitrogen atom to which they are attached form an optionallysubstituted 4-, 5-, 6- or 7-membered heterocyclic ring; R¹², R¹⁶, R¹⁹and R¹⁷ are as defined for R², R⁶, R⁹ and R⁷ respectively; R″ is a C₃₋₁₂alkylene group, which chain may be interrupted by one or moreheteroatoms and/or aromatic rings that are optionally substituted; and Xand X′ are independently selected from O, S and N(H).
 46. Theimmunoconjugate of claim 45, wherein D has the structure:

wherein n is 0 or
 1. 47. The immunoconjugate of claim 45, wherein D hasa structure selected from:

wherein R^(E) and R^(E″) are each independently selected from H orR^(D), wherein R^(D) is independently selected from R, CO₂R, COR, CHO,CO₂H, and halo; wherein Ar¹ and Ar² are each independently optionallysubstituted C₅₋₂₀ aryl; and wherein n is 0 or
 1. 48. The immunoconjugateof claim 41, wherein D is a pyrrolobenzodiazepine of Formula B:

wherein the horizontal wavy line indicates the covalent attachment siteto the linker; R^(V1) and R^(V2) are independently selected from H,methyl, ethyl, phenyl, fluoro-substituted phenyl, and C₅₋₆ heterocyclyl;and n is 0 or
 1. 49. The immunoconjugate of claim 41, wherein D is anemorubicin derivative.
 50. The immunoconjugate of claim 49, wherein Dhas a structure selected from:


51. The immunoconjugate of claim 41, wherein the linker is cleavable bya protease.
 52. The immunoconjugate of claim 51, wherein the linkercomprises a val-cit dipeptide or a Phe-homoLys dipeptide.
 53. Theimmunoconjugate of claim 41, wherein the linker is acid-labile.
 54. Theimmunoconjugate of claim 53, wherein the linker comprises hydrazone. 55.The immunoconjugate of claim 53 having the formula:

wherein S is a sulfur atom.
 56. The immunoconjugate of claim 46 having aformula selected from:


57. The immunoconjugate of claim 50 having a formula selected from:


58. The immunoconjugate of claim 41, wherein p ranges from 2-5.
 59. Apharmaceutical formulation comprising the antibody of claim 1 and apharmaceutically acceptable carrier.
 60. The pharmaceutical formulationof claim 59, further comprising an additional therapeutic agent.
 61. Amethod of treating an individual having an FcRH5-positive cancer, themethod comprising administering to the individual an effective amount ofthe antibody of claim
 1. 62. The method of claim 61, wherein theFcRH5-positive cancer is a B-cell proliferative disorder.
 63. The methodof claim 61, further comprising administering an additional therapeuticagent to the individual.
 64. A method of inhibiting proliferation of anFcRH5-positive cell, the method comprising exposing the cell to theantibody of claim 1 under conditions permissive for binding of theantibody and/or immunoconjugate to FcRH5 on the surface of the cell,thereby inhibiting proliferation of the cell.
 65. The method of claim64, wherein the cell is a B-cell.
 66. The antibody of claim 1, whereinthe antibody is conjugated to a label.
 67. The antibody of claim 66,wherein the label is a positron emitter.
 68. The antibody of claim 67,wherein the positron emitter is ⁸⁹Zr.
 69. A method of detecting humanFcRH5 in a biological sample comprising contacting the biological samplewith the anti-FcRH5 antibody of claim 1 under conditions permissive forbinding of the anti-FcRH5 antibody to a naturally occurring human FcRH5,and detecting whether a complex is formed between the anti-FcRH5antibody and a naturally occurring human FcRH5 in the biological sample.70. The method of claim 69, wherein the anti-FcRH5 antibody is anantibody comprising a) a heavy chain comprising a HVR-H1 comprising theamino acid sequence of SEQ ID NO:52, HVR-H2 comprising the amino acidsequence of SEQ ID NO:76, and HVR-H3 comprising the amino acid sequenceof SEQ ID NO:100; and/or b) a light chain comprising a HVR-L1 comprisingthe amino acid sequence of SEQ ID NO:4, HVR-L2 comprising the amino acidsequence of SEQ ID NO:16, and HVR-L3 comprising the amino acid sequenceof SEQ ID NO:28.
 71. The method of claim 69, wherein the biologicalsample is a blood sample.
 72. A method for detecting an FcRH5-positivecancer comprising (i) administering a labeled anti-FcRH5 antibody to asubject having or suspected of having a FcRH5-positive cancer, whereinthe labeled anti-FcRH5 antibody comprises the anti-FcRH5 antibody ofclaim 1, and (ii) detecting the labeled anti-FcRH5 antibody in thesubject, wherein detection of the labeled anti-FcRH5 antibody indicatesa FcRH5-positive cancer in the subject.
 73. The method of 72, whereinthe labeled anti-FcRH5 antibody comprises an anti-FcRH5 antibodyconjugated to a positron emitter.
 74. The method of claim 73, whereinthe positron emitter is ⁸⁹Zr.
 75. The method of claim 69, wherein theanti-FcRH5 antibody is an antibody comprising a) a heavy chaincomprising a HVR-H1 comprising the amino acid sequence of SEQ ID NO:55,HVR-H2 comprising the amino acid sequence of SEQ ID NO:79, and HVR-H3comprising the amino acid sequence of SEQ ID NO:103; and/or b) a lightchain comprising a HVR-L1 comprising the amino acid sequence of SEQ IDNO:7, HVR-L2 comprising the amino acid sequence of SEQ ID NO:19, andHVR-L3 comprising the amino acid sequence of SEQ ID NO:31.
 76. Apharmaceutical formulation comprising the immunoconjugate of claim 41and a pharmaceutically acceptable carrier.
 77. A method of treating anindividual having an FcRH5-positive cancer, the method comprisingadministering to the individual an effective amount of theimmunoconjugate of claim
 41. 78. A method of inhibiting proliferation ofan FcRH5-positive cell, the method comprising exposing the cell to theimmunoconjugate of claim 41 under conditions permissive for binding ofthe antibody and/or immunoconjugate to FcRH5 on the surface of the cell,thereby inhibiting proliferation of the cell.